Cleavable nucleic acid linkers for protein quantification ratioing

Abstract

The present disclosure concerns a Protein Quantification Reporter (PQR) linker which is capable of being cleaved during the translation of a messenger RNA to quantify a protein of interest. The PQR linker can encode a peptide of SEQ ID NO: 23 and have the nucleic acid sequence of SEQ ID NO: 25. The PQR linker is located in a nucleic acid molecule encoding a poly-protein between a reporter protein and the protein of interest. While the messenger RNA encoding the poly-protein is being translated, the presence of the PQR linker causes cleavage of the poly-protein and consequently the release at a stoichiometric ratio of the reporter protein and the protein of interest. The signal associated with the cleaved reporter protein can be measured to estimate or quantify the protein of interest.

Description

TECHNOLOGICAL FIELD

This disclosure relates to protein quantification ratioing using a cleavable nucleic acid linker intended to be located in a nucleic acid molecule encoding a protein of interest and a quantifiable protein marker.

BACKGROUND

The two most common methods for measuring absolute or relative protein amounts are protein assays and quantitative Western or immuno-blots. All methods for protein quantitation start with the isolation of large quantities of the cell type of interest due to the limited sensitivity and detection capabilities of these techniques, making them time consuming and laborious. The cellular resolution of these techniques is also limited because the isolated tissue is typically a heterogeneous population of cells that can include a wide range of cell types outside of a user's interest. These techniques are necessarily destructive processes as cells must be lysed to extract their protein content to be manipulated for the detection processes. Inaccuracies in quantitation using immuno-detection are further compounded by variabilities in antibody used to detect the protein, such as the avidity and affinity of the antibody, access of the antibody to the protein epitope, phosphorylation state of the protein, and cross-reactivities of the antibody. The use of a “housekeeping” protein for normalization is subject to the same limitations, as housekeeping protein quantification is still dependent on antibody detection, and differences across conditions, along with cellular heterogeneity can increase or decrease the housekeeping protein quantified without affecting the protein of interest (e.g., epithelial cells within neural tissue may not express a neural protein), leading to an inaccurate ratio between the protein of interest and the normalization control.

It would be highly desirable to be provided with a protein quantification method which would have heightened sensitivity and/or sensibility. It would also be desirable to be provided with a non-destructive protein quantification method that could allow, for example, live cell tracking. It would further be desirable to be provided with a protein quantification method that could be applied to a single cell. It would also be desirable to track and quantify protein production amounts over time in a cell by performing real-time measurements of protein production in single cells, at cellular or sub-cellular resolution.

BRIEF SUMMARY

The present disclosure concerns a Protein Quantitation Reporter (PQR) linker which is capable of being cleaved during the translation of a messenger RNA to quantify a protein of interest. The PQR linker can encode a peptide of SEQ ID NO: 23 and have the nucleic acid sequence of SEQ ID NO: 25. To quantify the protein of interest, the PQR linker is included in a nucleic acid molecule encoding a reporter protein and the protein of interest. In the nucleic acid molecule, the PQR linker is located between the reporter protein and the protein of interest. While the messenger RNA encoding the two proteins is being translated, the presence of the PQR linker forces a cleavage event between the two proteins and consequently causes the release of a stoichiometry ratio of the reporter protein and the protein of interest. The signal associated with the cleaved reporter protein can be measured to estimate or quantify the protein of interest.

According to a first aspect, the present disclosure provides a protein quantitation reporter linker molecule for quantifying a protein of interest in a host cell. Broadly, the protein quantitation reporter encodes a cleavable peptide having the amino acid sequence of SEQ ID NO: 23 and is a nucleic acid molecule having the nucleic acid sequence of SEQ ID NO: 25. The cleavable peptide has the amino acid sequence of SEQ ID NO: 23:

(SEQ ID NO: 23)
GSGX1X2X3X4X5X6X7X8X9LX10X11X12GDVEX13NPGP

in which X₁ is V or absent; X₂ is K or absent; X₃ is Q or absent; X₄ is T, C, A or absent; X₅ is L, T or E; X₆ is N or G; X₇ is F, Y or R; X₈ is D, A, G or S; X₉ is L or S; X₁₀ is K or L; X₁₁ is L, T or Q; X₁₂ is A or C and X₁₃ at position 21 is S or E.

The nucleic acid molecule has the nucleic acid sequence of SEQ ID NO: 25:

(SEQ ID NO: 25)
N1N2N3 N4N5N6 N7N8N9 N10N11N12 N13N14N15 N16N17
N18 N19N20N21 N22N23N24 N25N26N27N28N29N30 N31
N32N33 N34N35N36 N37N38N39 N40N41N42 N43N44N45
N46N47N48 N49N50N51 N52N53N54N55N56N57 N58N59
N60 N61N62N63 AAY CCN64 GGA CCN65

in which N₁ to N₆₃ are any nucleic acid capable of forming codons encoding GSGX₁X₂X₃X₄X₅X₆X₇X₈X₉LX₁₀X₁₁X₁₂GDVEX₁₃ of SEQ ID NO: 23, N₆₄ is T or U and N₆₅ is T or U; and at least 50% of the codons encoding X₁X₂X₃X₄X₅X₆X₇X₈X₉LX₁₀X₁₁X₁₂GDVEX₁₃ of SEQ ID NO: 23 correspond to the codons least favored in the host cell. In an embodiment, the cleavable peptide has the amino acid sequence of SEQ ID NO: 24:

(SEQ ID NO: 24)
GSGX14X15X16X17X18X19LX20X21X22GDVEENPGP

in which X₁₄ is A or absent; X₁₅ is E or T; X₁₆ is G or N; X₁₇ is R or F; X₁₈ is G or S; X₁₉ is S or L; X₂₀ is L or K; X₂₁ is T or Q and X₂₂ is C or A. In another embodiment, the protein quantitation reporter linker molecule is a deoxyribonucleic (DNA) molecule. In still a further embodiment, the protein quantitation reporter linker molecule has a nucleic acid sequence of SEQ ID NO: 26:

(SEQ ID NO: 26)
N1N2N3 N4N5N6 N7N8N9 N10N11N12 N13N14N15 N16N17
N18 N19N20N21 N22N23N24 N25N26N27N28N29N30 N31N32
N33N34N35N36 N37N38N39 N40N41N42 N43N44N45 N46N47
N48 N49N50N51 N52N53N54N55N56N57 N58N59N60
N61N62N63 AAY CCT GGA CCT

in which N₁ to N₆₃ are any nucleic acid capable of forming codons encoding GSGX₁X₂X₃X₄X₅X₆X₇X₈X₉LX₁₀X₁₁X₁₂GDVEX₁₃of SEQ ID NO: 23. In yet another embodiment, in the nucleic acid encoding the protein quantitation reporter linker, at least 80% of the codons encoding X₁X₂X₃X₄X₅X₆X₇X₈X₉LX₁₀X₁₁X₁₂GDVEX₁₃ of SEQ ID NO: 23 correspond to the codons least favored in the host cell.

In a second aspect, the present disclosure provides a vector for quantifying a protein of interest in a host cell, said vector comprising a first nucleic acid molecule encoding the protein quantitation reporter linker described herein. In an embodiment, the vector further comprises a second nucleic acid molecule encoding a reporter protein operatively linked to the first nucleic acid molecule so that the first nucleic acid molecule and second nucleic acid molecule are transcribed as a single messenger RNA transcript from the vector. In another embodiment, the vector further comprises a third nucleic acid molecule encoding a protein of interest operatively linked to the first nucleic acid molecule so that the first nucleic acid molecule and the third nucleic acid molecule are transcribed as a single messenger RNA transcript from the vector. In still another embodiment, the vector further comprises a second nucleic acid molecule encoding a reporter protein and a third nucleic acid molecule encoding a protein of interest, wherein the second nucleic acid molecule and the third nucleic acid molecule are operatively linked to the first nucleic acid molecule and wherein the first nucleic acid molecule is located between the second nucleic acid molecule and the third nucleic acid molecule, so that the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule are transcribed as a single messenger RNA transcript from the vector. In yet another embodiment, the second nucleic acid molecule is upstream of the third nucleic acid molecule or downstream of the third nucleic acid molecule. In still a further embodiment, the reporter protein is selected from the group consisting of a fluorescent protein, an antibiotic-resistance protein, an immunoglobulin protein, an ion channel, a transcription factor, a ribosomal protein, an enzyme and a receptor. In yet a further embodiment, the fluorescent protein is selected from the group consisting of a green-fluorescent protein (GFP), a red fluorescent protein (RFP), a yellow fluorescent protein (YFP), a blue fluorescent protein (BFP) and a cyan fluorescent protein (CFP).

According to a third aspect, the present disclosure provides a kit for quantifying a protein of interest in a host cell, said kit comprising the vector of described herein and instructions for using the vector to quantify the protein of interest.

According to a fourth aspect, the present disclosure provides a transgenic host cell comprising the vector described herein.

According to a fifth aspect, the present disclosure provides a transgenic host cell comprising (i) a first nucleic acid molecule encoding the protein quantitation reporter linker described herein, (ii) a second nucleic acid molecule encoding a reporter protein and (iii) a third nucleic acid molecule encoding a protein of interest, wherein the second nucleic acid molecule and the third nucleic acid molecule are operatively linked to the first nucleic acid molecule and wherein the first nucleic acid molecule is located between the second nucleic acid molecule and the third nucleic acid molecule, so that the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule are transcribed as a single messenger RNA transcript. In an embodiment, the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule are integrated in the genome of the host cell.

According to a sixth aspect, the present disclosure provides a host comprising the transgenic host cell described herein.

According to a seventh aspect, the present disclosure provides a method for quantifying a protein of interest in a host or a host cell. Broadly the method comprises (i) expressing the vector described herein in the host or the host cell so as to cause the generation of a nucleic acid transcript encoding a poly-protein, wherein the poly-protein comprises the protein of interest, a cleavable peptide and a reporter protein and wherein the cleavable peptide can be cleaved during the translation of the nucleic acid transcript to generate a cleaved reporter protein and (ii) measuring a signal associated with the cleaved reporter protein to quantify the protein of interest. In an embodiment, the reporter protein is a fluorescent protein and step (ii) further comprises determining the fluorescence associated with the cleaved reporter protein. In still another embodiment, the host cell is a living cell. In yet another embodiment, the host cell is a single cell.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus generally described the nature of the invention, reference will now be made to the accompanying drawings, showing by way of illustration, a preferred embodiment thereof, and in which:

FIG. 1A illustrates that Protein Quantitation Ratioing (PQR) can determine relative protein concentration in single living cells. Stoichiometric protein translation can quantitate protein amounts. Insertion of a Protein Quantitation Reporter (PQR) between a fluorescent reporter (GFP) and a gene of interest creates a polycistronic mRNA for co-transcription and co-translation of GFP and the gene of interest. The PQR construct allows for one molecule of GFP (having an additional) C-terminal tail consisting of residues 6 to 18 of SEQ ID NO: 1 for example) to be synthesized for every one proportional to the concentration of GFP, then the fluorescence output of GFP is directly proportional to the concentration of GFP, then the fluorescence intensity of a cell can be used to quantitate the concentration of the protein of interest.

FIG. 1B illustrates the linear relationships between fluorescence output, fluorescent protein concentration, and protein of interest concentration allowing for Protein Quantitation Ratioing. Because the fluorescence output of GFP is directly proportional to its concentration (top panel and FIG. 1C), then using a PQR will produce a stoichiometric ratio between GFP and the protein of interest (middle panel), therefore enabling the fluorescence intensity of GFP to be used as a measure of the protein of interest concentration (bottom panel). Any (linear) differences in post-translational processing, maturation, or insertion rates of the protein of interest or GFP will change the slope of the relationship (dotted gray lines). For example, if at steady-state there are 11 functional molecules of a Shaker K+ channel for every 41 functional molecules of GFP, the relationship will still be linear. Importantly, protein concentrations is predominantly controlled by translation, with very small contribution from protein stability and degradation.

FIG. 1C shows that fluorescence intensity of GFP increases linearly as a function of its concentration over five orders of magnitude. Purified GFP was imaged using standard fluorescence microscopy. Pixel intensities are plotted in arbitrary units (a.u.) in log₁₀. Coefficient of determination, R² values from a simple linear regression model were calculated from the averages of five experiments. Error bars standard deviations.

FIG. 1D shows that the fluorescence intensity of RFP increases linearly as a function of its concentration over five orders of magnitude. Purified RFP was imaged using standard fluorescence microscopy. Pixel intensities are plotted in arbitrary units (a.u.) in log₁₀. Coefficient of determination, R² values from a simple linear regression model were calculated from the averages of five experiments. Error bars are standard deviations.

FIG. 2A illustrates that wild-type viral cis-acting hydrolase element (CHYSEL) sequences produce un-separated fusion proteins; PQR sequences produce reliable separation of separation efficiency using immunoblots (representative examples shown) and stoichiometric production of proteins using quantitative imagining. Anti-GFP antibody was used to detect GFP (middle blots) versus fusion product of unseparated RFP and GFP (top blots). Anti-Actin (bottom blots) was used to normalize pixel intensities of fusion product (numbers underneath top blots). We added gylcine and serine N-terminus linkers to all synthesized CHYSEL sequences, for example on the 2A-like sequence from Thosea asigna virus (T2A). Wildtype T2A viral codon usage or codon optimization produces fusion protein production, whereas codon de-optimization enhances separation efficiency. Separation efficiency for each CHYSEL construct was tested using immunoblotting of RFP-CHYSEL-mCD8::GFP constructs transfected into Drosophila S2 cells for T2A-derived sequences. Manipulation of the T2A peptide sequence by adding glycine and serine linkers still produced a large fraction of fusion protein (arrowhead in Lane 2, “viral” T2A). When we turned to manipulating codon sequence usage, we found that codon optimization produced equivalent or worse amounts of fusion products (arrowhead in Lane 5, T2A variant 3, 100% codon optimized) compared to the viral CHYSEL sequence, along with diminished amounts of separated mCD8::GFP. Codon de-optimization of specific amino acids reduced the proportion of fusion product (Lane 3, T2A variant 1 is 60% codon de-optimized and Land 4, T2A variant is 45% codon de-optimized). T2A variant 2, 45% codon de-optimization (asterisk), produced close to the background levels of the untransfected S2 cells lane. T2A mutant constructs (Lane 6) that produced fusion products were use as positive controls. All codon percentage change numbers do not include the glycine serine linker codons, which were required in all constructs (including “viral” sequences) to avoid large amounts of fusion products within the proteasome. Thus, using viral CHYSEL sequences will not work as Protein Quantitation Reporters, as these sequences leave a large fraction of uncleaved fusion protein product (arrowheads) that will contaminate any results of quantitation, and any experiments where fusion products are undesirable.

FIG. 2B illustrates that wild-type viral cis-acting hydrolase element (CHYSEL) sequences produces un-separated fusion proteins: PQR sequences produce reliable separation of proteins. We modified and synthesized different viral CHYSEL sequences to screen for separation efficiency using immunoblots (representative examples shown) and stoichiometric production of proteins using quantitative imaging. Anti-GFP antibody was used to detect GFP (middle blots versus fusion product of unseparated RFP and GFP (top blots). Anti-Actin (bottom blots) was used to normalize pixel intensities of fusion product (numbers underneath top blots). We added glycine and serine N-terminus linkers to all synthesized CHYSEL sequences, for example on the 2A sequences from Porcine teschovirus-1 (P2A). Codon de-optimization of specific CHYSEL residues produces reliable separation of proteins. We used HEK293 cells to test codon de-optimization of different CHYSEL residues using RFP—CHYSEL—GFP constructs derived from P2A sequences. We found that ˜50% codon de-optimization of sequences (Lane 6, P2A variant 3), without altering the final four codons, allows for greatest separation efficiency. P2A variant 4, with the last 4 codons de-optimized (Lane 4) produced similar amounts of fusion product as the positive control (Lane 3), with negligible amounts of unseparated GFP (middle blot). P2A variants 1, 2, and 3, changing 100%, 80%, and 50%, respectively, of the codons (except for the last 4 codons) produced decreasing amounts of fusion product and increasing amounts of separated GFP. All codon percentage change numbers do not include the glycine serine linker codons, which were required in all constructs (including “viral” sequences) to avoid large amounts of fusion products within the proteasome. Thus, using viral CHYSEL sequences will not work as Protein Quantitation Reporters, as these sequences leave a large fraction of uncleaved fusion protein product (arrowheads) that will contaminate any results of quantitation, and any experiments where fusion products are undesirable.

FIG. 3A shows that red and green fluorescence intensities of HEK293 cells expressing a fusion protein of GFP and RFP (GFP::RFP) were linearly correlated with a coefficient of determination, R²=0.74(n=74 cells, P<0.001). Fluorescence values are in arbitrary units.

FIG. 3B shows that co-transfection of GFP and RFP produced a weak correlation between fluorescence intensities (n=59 cells, P<0.001).

FIG. 3C shows that insertion of a PQR between GFP and RFP produces red and green fluorescence intensities that were linearly correlated. R² values for GFP-PQR-RFP (n=77) was not significantly different from the fusion protein data (P>0.05).

FIG. 3D shows that insertion of a PQR between GFP and RFP produces red and green fluorescence intensities that were linearly correlated. R² values for RFP-PQR-GFP (n=77) was not significantly different from the fusion protein data (P>0.05).

FIG. 3E shows whole cell patch clamp recordings performed on HEK293 cells.

FIG. 3F shows an I-V curve generated using +10 voltage steps during a whole cell patch clamp recordings performed on HEK293 cells.

FIG. 3G shows sample micrographs of ShakerGFP-PQR-RFP-transfected HEK293 used to measure the GFP fluorescence intensity. The GFP signal (left panel) is localized to the plasma membrane whereas the RFP signal (right panel) is cytoplasmic. Scale bar is 25 μm.

FIG. 3H shows that K⁺ channel current density was linearly correlated with green fluorescence intensity in cells expressing the Shaker K⁺ channel fused to GFP, with a coefficient of determination. R²+0.73 (n=28 cells, P<0.001). Steady-state current was measured at +mV and current density (pA/pF) was calculated using the membrane capacitance.

FIG. 3I shows that red fluorescence intensities were correlated with K³⁰ channel current density in cells expressing ShakerGFP-PQR-RFP. R² values for current density to RFP, and GFP to RFP were not significantly different from the current density to GFP positive control data (P>0.05); see also FIGS. 7 and 9. These correlations were not due to unseparated RFP fusion products since green fluorescence was restricted to the membrane, and red fluorescence remained cytoplasmic (images in 3G). All fluorescence intensities are plotted in arbitrary units (a.u.).

FIG. 3J shows that red fluorescence intensities were correlated with green fluorescence in cells expressing ShakerGFP-PQR-RFP. R² values for current density to RFP, and GFP to RFP were not significantly different from the current density to GFP positive control data (P>0.05); see also FIGS. 7 and 9. These correlations were not due to unseparated RFP fusion products since green fluorescence was restricted to the membrane, and red fluorescence remained cytoplasmic (images in 3G). All fluorescence intensities are plotted in arbitrary units (a.u.).

FIG. 4A illustrates that PQR can relate cellular phenotype as a function of protein concentration. PQR can detect cyclic increases in protein concentration over time. RFP-PQR-PER::YFP was used to quantitate changes in PER transcription factor levels in single neurons in the animal. An image of the Drosophila brain is shown with RFP and PER::YFP expression restricted to the small lateral ventral neurons (dotted box and right panels) using Per-Gal4 to drive UAS—RFP-PQR-PER::YFP. Red fluorescence within the neurons remained in the cytoplasm, and yellow fluorescence was peri-nuclear. Scale bars are 100 μm (left panel) and 10 μm (right panels).

FIG. 4B shows that red fluorescence (full line) increased cyclically in neurons over days. Flies were entrained on a 12 hour light-dark cycle and red and yellow fluorescence intensities were measured within single neurons at zeitgeber time 0 (sun symbol) and 12 (moon symbol) (n=6 cells/6 animals/time point). Yellow fluorescence (dashed line) intensities cycles every 24 hours without accumulating beyond a fixed value, reflecting the rapid lifetime of PER. Red fluorescence (fill line) intensities were also cyclical, but gradually increased over several days, reflecting the integrated amount of PER produced over time. Error bars are S.E.M. See also FIG. 10.

FIG. 4C shows PQR in single living neurons being used to quantitatively relate dendritic complexity with Cut protein levels. Dendritic complexity of Drosophila da neurons is regulated by the transcription factor Cut. Wild-type class I da Neurons (left panel) have relatively simple dendritic arbors. Expression of RFPnls-PQR-cut within class I neurons increases dendritic branch number and total dendritic branch length (middle and right panels). Red fluorescence within the nucleus (inset in middle and right panels) reflecting Cut protein levels indicates that Cut controls dendritic growth in a concentration dependent manner. Posterior is up and dorsal to the right in all three panels. Scale bar is 30 μm.

FIG. 4D shows that dendritic complexity is logarithmically dependent on Cut protein concentration. The average number of dendritic branch terminals is indicated by the solid grey line (+1 S.D., dashed lines).

FIG. 4E shows that dendritic complexity is logarithmically dependent on Cut protein concentration. The total dendritic length in wild-type neurons is indicated by the solid grey line (+1 S.D., dashed lines).

FIG. 5A illustrates that PQRs can be inserted into any genomic locus to quantitate endogenous protein levels. Insertion of a PQR before the final stop codon of the edogenous gene maintains the mRNA production fidelity and the 3′ untranslated region (UTR) for all isoforms of the mRNA with the PQR. A site-specific DNA double-strand break is created using the CRISPR-Cas9 system. The break is repaired by the cell using homologous recombination, and in the presence with the PQR edited version. Shaded nucleotide sequences represent genomic sequencing results of an edited mouse RPL13A gene with a PQR-RFP insertion.

FIG. 5B shows that targeted genome editing allows for insertion of a PQR into the human genome. A repair template and guide RNA for CRISPER-Cas9 was designed for the RPL13A gene in human. RPL13A gene edited with PQR produced RFP. PQR insertion was verified using genomic PCR genotyping with primer pairs (see Table 4B) that spanned PQR and outside the homology arms, followed by genomic sequencing. Scale bars are 100 μm.

FIG. 5C shows that targets genome editing allows for insertion of a PQR into the drosophila genome. A repair template and guide RNA for CRISPR-Cas9 was designed for the RPL13A gene in drosophila. RPL13A gene edited with PQR produced RFP. PQR insertion was verified using genomic PCR genotyping with primer pairs (see Table 4B) that spanned PQR and outside the homology arms, followed by genomic sequencing. Scale bars are 100 μm.

FIG. 5D shows that targets genomes editing allows for insertion of a PQR into the mouse genome. A repair template and guide RNA for CRISPER-Cas9 was designed for the RPL13A gene in mouse. RPL13A gene edited with PQR produced RFP or BFP with a nuclear localization signal (BFPnls). PQR insertion was verified using genomic PCR genotyping with primer pairs (see Table 4B) that spanned PQR and outside the homology arms, followed by genomic sequencing. Scale bars are 100 μm.

FIG. 6A illustrates protein quantification in single cells. Fluorescence intensity of single cells with a PQR knock-in is measured and cells are then lysed for total RNA extraction and single cell quantitative PCR.

FIG. 6B demonstrates that the frequency distribution of RPL13A mRNA amounts measured from single HEK293 cells exhibits moderate expression of the RPL13A gene. Quantitative PCR of RPL13A mRNA specifically containing PQR constructs was performed to avoid variability due to heterozygosity or polyploidy of the cells.

FIG. 6C shows that RFP fluorescence intensities (in arbitrary units) from single RPL13A-PQR-RFP knock-in cells exhibit a moderate distribution.

FIG. 6D shows that RFP fluorescence intensities (in arbitrary units) from single RPL13A-PQR-RFP knock-in cells exhibit a weak linear correlation to mRNA amounts (n=22. R²=0.03).

FIG. 6E shows that the endogenous immunoglobulin kappa light chain (lgK) locus is edited to insert a PQR-GFP reporter at the end of the constant region in 22c10 mouse hybridoma cells (top panel). The correct insertion is verified by PCR primer pairs (see Table 4B) that lie within and outside of the locus (arrows, bottom right panel). 22c10 hybridoma cells produce green fluorescence (bottom left panel) after insertion of a PQR-GFP into the endogenous lgK locus. Scale bar is 25 μm. Representative PCR genotyping results show the expected size in the CRISPR-Cas9 transfected cells.

FIG. 6F shows that frequency distribution of lgK mRNA amounts measured from single 22c10 cells exhibits a broad range and high level of mRNA and protein expression.

FIG. 6G shows that frequency distribution of PQR-GFP fluorescence intensities measured from single 22c10 cells exhibits the broad range and high level of mRNA and protein expression.

FIG. 6H shows that the lgK protein expression was not strongly correlated with its mRNA amounts; see also FIG. 9.

FIG. 7A illustrates the statistical analysis of R² values from experiments, related to FIG. 3. How accurate are our R² values for each experiment? We tested the null hypothesis that the experimental variables were independent of each other and that the true R² value was 0. We used the permutation test to obtain a P value on the likelihood of obtaining out R² value by randomly shuffling the data and calculating a new R² value, repeated for one million runs. A representative example is shown for the frequency distribution of randomly permuted R² values derived from the ShakerGFP current density versus RFP data. The experimental R2 value was 0.55(n=28 cells) and was highly significant from the average randomly permuted R² value, with three S.D.=0.20.

FIG. 7B shows Bootstrap of positive control fusion protein data being used to compare between experiments. Our experimentally derived R² values were not equal to 1 most likely due to non-linear differences in protein kinetics between the two proteins, intramolecular interactions for the fusion proteins, and experimental precision. Given that our R² values for each experiment were significantly greater than random covariance (i.e., the true R2 is not close to 0) (a) but less than a perfect R²=1, we needed a method to quantitatively compare R² values between experiments. We used the data from the fusion protein experiments as positive controls to compare the R¹ values among other conditions. We used the bootstrap method to generate a 95% confidence interval for the true R² value of the positive controls. We randomly chose 80% of the positive control data points to calculate a new R² value and repeated this for ten million runs, and used these simulated R² values to obtain upper and lower estimates of the positive control R² values. A representative example is shown for the frequency distribution of R² values calculated from randomly choosing 90% of the GFP::RFP data, performed ten million times. R² values for the RFP-PQR-GFP and GFP-PQR-RFP fell within the 95% confidence interval, whereas the R² value for co-transfection of GFP and RFP did not.

FIG. 8A illustrates that Protein Quantitation Rationing can be used with multiple microscopy methods and multiple fluorophores within different subcellular compartments, related to FIG. 3. Image analysis of HEK293 cells expressing YFPmito-PQR-CFPnls-PQR-RFP demonstrates linear correlations between RFP and CFP fluorescence intensities within different subcellular compartments. We used mitochondrial (mito) and nuclear (nls) localization signals on YFP and CFP, respectively, with RFP remaining cytosolic. Fluorescence intensities within the nucleus compared to cytoplasm were consistently the most highly correlated.

FIG. 8B shows an image analysis of HEK293 cells expressing YFPmito-PQR-CFPnls-PQR-RFP demonstrating linear correlations between CFP and YFP fluorescence intensities within different subcellular compartments. We used mitochondrial (mito) and nuclear (nls) localization signals on YFP and CFP, respectively, with RFP remaining cytosolic.

FIG. 8C shows an image analysis of HEK293 cells expressing YFPmito-PQR-CFPnls-PQR-RFP demonstrating linear correlations between RFP and YFP fluorescence intensities within difference subcellular compartments. We used mitochondrial (mito) and nuclear (nls) localization signals on YFP and CFP, respectively, with RFP remaining cytosolic.

FIG. 8D shows that different fluorescence microscopy methods can be used with PQR. Fluorescence output remains linear between different excitation sources and microscopy methods. HEK293 cells expressing ShakerGFP-PQR-REP were imaged using a spinning disk confocal microscope. Red Fluorescence intensities were highly correlated between excitation methods using a Kr 568nm laser and Hg lamp with R²=0.90(n=46 cells, P<0.001). All excitation methods, fluorophores, and microscopy methods tested produces R² values ≥0.90 (representative example shown).

FIG. 8E shows that HEK293 cells transfected with RFP-PQR-ShakerGFP did not display inappropriate localization of the Shaker K⁺ channel, despite the addition of the remaining proline at the N-terminus of the transmembrane channel due to separation of the PQR peptide. These results demonstrate that our PQRs separate robustly regardless of the order or size of the upstream or downstream genes, and that the addition of the N-terminus or C-terminus PQR tags do not interfere with protein function or trafficking.

FIG. 9A illustrates that RNA and protein quantitation of both Rpl13a and lgk simultaneously in single living cells using a double knock-in, related to FIG. 6. POQR-GFPnls was inserted into the mouse Rpl13a locus on chromosome 7 using CRISPR-Cas9 genome editing, while PQR-RFP was inserted into the lgk locus on chromosome 6.

FIG. 9B shows that protein expression minimally co-varies with mRNA expression for both genes. The number of RNA transcripts for both genes (x-axis) were measured using qPCR of single cells and green and red fluorescence intensities were measured using a standard fluorescence microscope. Black lines connect the data from the same cell.

FIG. 9C shows a representative example of a double knock-in 22c10 cell expressing GFP in the nucleus and RFP in the cytoplasm, which corresponds to its RPL13A and lgk protein amounts, respectively. Scale bar is 20 μm.

FIG. 10A illustrates that PQR can accurately measure relative protein amounts, related to FIG. 4. Simulation of cells expressing a protein of interest with a PQR. Protein of interest data was modeled from human, mouse, and yeast proteome datasets, and each cell had a random ON and OFF rate, abundance, and lifetime for the protein of interest. PQR-GFP had identical ON rates as well as protein production amounts. When the PQR fluorophore has different kinetics than the protein of interest, absolute protein quantification is inaccurate due to differences in protein turnover (Cell 1, black arrows). By measuring the relative amounts of GFP and comparing this to the relative amounts of the protein of interest, relative protein quantification is highly accurate even for cells with widely varying protein expression (Cell 2, note protein of abundance and kinetics).

FIG. 10B shows a representative example of histogram of relative differences between GFP and protein of interest measurements over 1,000 trials for the protein in (10A). Relative difference greater than 1 indicate that the PQR-GFP measurement would overestimates of the true protein of interest amount. PQR has >90% accuracy at >85% pf data points across tens of thousands of protein simulations.

FIG. 10C shows PQR measurements in Drosophila neurons controlling circadian rhythms in phase cycling with PER protein production, at an arbitrary animal age. Small lateral ventral neurons in the Drosophila brain expressing Per-Gal4 to drive UAS—RFP-PQR-PER::YFP were analyzed for yellow (dashed line) and red (full line) fluorescence. Red fluorescence intensity values cycled in phase with yellow fluorescence at Day 5 and 6 when measured with a lower acquisition setting than in FIG. 4. Error bars are S.E.M.

FIG. 11A illustrates the full immuno-blots of different CHYSEL variants and PQR constructs used, related to Experimental Procedures. Screening through different CHYSEL variants from different viruses and within different model ogranism cell lines produced PQR CHYSEL variants with consistent separation of the upstream and downstream proteins. Different cell lines were performed to detect the presence of GFP fused to RFP, and GFP alone. Uncropped blot of T2A-derived sequence variant from FIG. 2. Variant chosen for PQR produces more cleaved protein (lower arrowheads for mCD8::GFP) and less fusion product (upper arrowheads for RFP-T2A-mCD8::GFP). than other CHYSEL peptides. Asterisk denotes the variant chosen as the PQR sequence for experiments (see Example 2 and FIG. 2 legend).

FIG. 11B shows screening of different CHYSEL variants from different viruses and within different model organism cell lines producing PQR CHYSEL variants with consistent separation of the upstream and downstream proteins. Different cell lines were transfected with GFP-CHYSEL-RFP, and Western blots using anti-GFP antibody were performed to detect the presence of GFP fused to RFP, and GFP alone. Uncropped blot of P2A-derived sequence variant from FIG. 2. Variant chosen for PQR produces more cleaved protein (lower arrowheads for GFP) and less fusion product (upper arrowheads for GFP-P2A-RFP) than other CHYSEL peptides. Asterisk denotes the variant chosen as the PQR sequence for experiments (see Example 2 and FIG. 2 legend).

FIG. 11C shows screening of different CHYSEL variants from different viruses and within different model organism cell lines producing PQR CHYSEL variants with consistent separation of the upstream and downstream proteins. Different cell lines were transfected with GFP-CHYSEL-RFP, and Western blots using anti-GFP antibody were performed to detect the presence of GFP fused to RFP, and GFP alone, PQR sequences outperform all other versions of CHYSEL peptides including both short (19 amino acids) and long (30 amino acids) viral sequences.

FIG. 11D shows uncropped immuno-blots on all PQR constructs used in experiments to demonstrate the absence of any fusion protein products that might confound our analysis, HEK293 cells expressing fusion protein GFP::RFP. GFP and RFP plasmids co-transfected. GFP-PQR-RFP, and RFP-PQR-GFP were lysed 5 days after transfection. Collected protein content was analyzed using anti-GFP antibody. Cells that were transfected with either GFP-PQR-RFP or RFP-PQE-GFP produced low or undetectable amounts of fusion product (upper arrow).

FIG. 11E shows uncropped immuno-blots on all PQR constructs used in experiments to demonstrate the absence of any fusion protein products that might confound our analysis, HEK293 cells expressing ShakerGFP-PQR-RFP were analyzed in Western blots using anti-RFP antibody, and no fusion product was detected.

FIG. 11F shows uncropped immuno-blots on all PQR constructs used in experiments to demonstrate the absence of any fusion protein products that might confound our analysis. Kc cells expressing RFP-PQR-PER::YFP and RFPnls-PQR-cut were analyzed in immuno-blots using anti-RFP antibody. No fusion proteins were produced from either of the two PQR constructs.

FIG. 12A illustrates that the knock-in of PQR into endogenous loci does not produce fusion proteins nor significantly after the mRNA expression, related to Experimental Procedures. Genome-edited HEK293 cells were lysed and protein content was analyzed using immunoblots against RFP. RFP protein bands were observed, but no fusion protein products were detected.

FIG. 12B illustrates that the knock-in of PQR into endogenous loci does not produce fusion proteins nor significantly alter the mRNA expression, related to Experimental Procedures. Genome-edited N2A cells were lysed and protein content was analyzed using immunoblots against RFP. RFP protein bands were observed, but no fusion protein products were detected.

FIG. 12C shows a quantitative real-time PCR analysis of knock-in PQR cells demonstrating that relative levels of mRNA of the PQR-edited gene are not changed. PQR-specific mRNA was measured and normalized to GAPDH mRNA levels and compared as fold changes from the levels in untransfected cells. In 22c10, qPCR experiments were performed in duplicate with technical replicates in duplicate on the lgk loci.

FIG. 12D shows a quantitative real-time PCR analysis of knock-in PQR cells demonstrating that relative levels of mRNA of the PQR-edited gene are not changes. PQR-specific mRNA was measured and normalized to GAPDH mRNA levels and compared as fold changes from the levels in untransfected cells. in N2A cells, experiments were performed in quadruplicate with technical replicates in duplicate on the RPL13A locus.

FIG. 12E shows a quantitative real-time PCR analysis of knock-in PQR cells demonstrating that relative levels of mRNA of the PQR-edited gene are not changed. PQR-specific mRNA was measured and normalized to GAPDH mRNA levels and compared as fold changes from the levels in untransfected cells. In HEK293T (293T) cells, qPCR experiments were performed in duplicate with technical replicates in duplicate on the RPL13A loci.

FIG. 13A illustrates the importance of codon de-optimization in the PQR. Western blot results provided as the amount of the fusion protein (RFP-GFP), of the cleaved protein (GFP) and a house-keeping protein (actin) in function of DNA sequence used. “Viral” P2A, variations 1, 2, 3, and 4, correspond to SEQ ID NOs: 5, 6. 10, 11, and 12, respectively.

FIG. 13B shows western blot results provided as the amount of the fusion protein (RFP-GFP), of the cleaved protein (GFP) and a house-keeping protein (actin) in function of DNA sequence used. “Viral” T2A, variations 1, 2, and 3, correspond to SEQ ID NOs: 8, 19, 15, and 16, respectively.

FIG. 13C shows a quantification of western blot results from 13A.

FIG. 13D shows a quantification of western blot results from 13B.

FIG. 14A illustrates modifications of the Glycine-Serine-Glycine (GSG) in CHYSEL peptides. The glycine receptor was expressed with GFP using F2A peptide with a GSG modification (i.e., addition). HEK 293 cells expressing the glycine receptor with the GSG modification were imaged (top panel) and whole cell patch clamp electrophysiology was performed (bottom panel). Cells expressing the gylcine receptor with the GSG modification displayed uniform GFP fluorescent throughout the cell indicating appropriate cleavage of the two proteins, and produces large glycine mediated currents. Scale bar is 10 μm.

FIG. 14B illustrates modifications of the Glycine-Serine-Glycine (GSG) in CHYSEL peptides. The glycine receptor was expressed without the GSG modification. HEK 293 cells expressing the glycine receptor and GFP without the GSG modification to the CHYSEL peptide contained GFP puncta throughout the cell where the un-cleaved fusion protein was degraded and sequestered within multiple inclusion bodies (top panel, yellow arrows), and produced no detectable Glycine current (bottom panel). Scale bar is 10 μm.

FIG. 15A illustrates similar linear correlations between fluorophores separated using F2A peptide with a GSG modification. Insertion of a GSG-F2A peptide between GFP and RFP produces red and green fluorescence intensities that were linearly correlated. R² values for GFP-GSG-F2A-RFP were similar to those obtained using P2A and T2A constructs.

FIG. 15B illustrates similar linear correlations between fluorophores separated using F2A peptide with a GSG modification. Insertion of a GSG-F2A peptide between GFP and RFP produces red and green fluorescence intensities that were linearly correlated. R² values for GFP-GSG-F2A-RFP were similar to those obtained using P2A and T2A constructs.

FIG 16A illustrates the design of a RPL13A-PQR-RFPnols knock-in Drosophila. A knock-in Drosophila expressing the nucleolar RFP (RFPnols) reporter from the endogenous RPL13A locus. Shaded nucleotide sequences represent genomic sequencing results of an edited Drosophila RPL13A gene with a PQR-RFPnols insertion.

FIG. 16B shows a heterozygous RPL13A-PQR-RFPnols fly verified using genomic PCR genoptyping with primer pairs (see Table 4B) that spanned PQR-RFPnols and outside of the homology arms. For primers spanning outside of homology arms (see Table 4B), PQR-RFPnols allele can produce PCR product size of 3.9 kb (upper arrow), while WT allele resulted in PCR product size of 3 kb (middle arrow). When primer set A or B was used for genotyping, only the knock-in fly produced PCR amplicon of kb 1.8 kb (bottom arrow).

FIG. 16C shows fluorescence micrographs of red nuclei observed from all the cells in embryos. Scale bars are 50 μm.

FIG. 16D shows fluorescence micrographs of red nuclei observed from all the cells in larval body walls. Dopamine Neurons labeled in Green were used to visually delineate each segment of an entire larval body. Scale bars are 50 μm.

DETAILED DESCRIPTION

The present disclosure concerns a method of quantifying a protein of interest as well as tools associated thereto. The method relies on the use of a Protein Quantitation Reporter (PQR) linker which is capable of being cleaved during the protein translation of a messenger RNA to quantify a protein of interest. The PQR linker is a nucleic acid molecule encoding a peptide linker located between a reporter protein and the protein of interest. While the messenger RNA encoding the poly-protein is being translated, the PQR peptide linker is cleaved which causes the release, in a stoichiometric ratio, of the reporter protein and the protein of interest. The signal associated with the cleaved reporter protein can be measured to estimate or quantify the protein of interest.

Protein Quantitation Reporter (PQR) Linker

In its broadest embodiment, the PQR linker encodes a cleavable peptide located between two proteins. The PQR linker is intended to be cleaved, during protein translation to produce a stoichiometric ratio of a reporter protein and the protein of interest. In an embodiment, the PQR linker is cleaved at a frequency of at least 95% and, in some further embodiment, the PQR linker is cleaved at a frequency of at least 96%, 97%, 98% or 99%. The signal associated to reporter protein is thus proportional to the amount of protein of interest and is used to determine the relative amount of the protein of interest within the cell.

In an embodiment, the PQR linker encodes a modified cis-acting hydrolase element (CHYSEL) peptide to which a GSG tripeptide has been added to the amino (i.e., NH₂) terminus. The CHYSEL peptide includes, at its carboxyl end, a “PGP” tri-peptide. In such embodiment, the PQR linker encodes a cleavable peptide which can be cleaved between the carboxy's penultimate glycine and the carboxy's ultimate proline of the CHYSEL peptide. CHYSEL peptides, also known as “2A” and “2A-like” peptides, come from a broad range of Group IV, positive-sense single stranded RNA viruses such as the in Picornaviridae family including the Aphthoviruses: Equine rhinitis A virus (expressing the E2A peptide), the Foot-and-mouth disease virus (expressing the F2A peptide), and also the Teschovirus: Porcine teschovirus (expressing the P2A peptide). CHYSEL peptides of the 2A-like variety can come from Alphapermutotetraviruses in the Permutotetraviridae family, such as the Thosea asigna (expressing the T2A peptide), or from the Dicistroviridae family such as Drosophila C virus (expressing the D2A peptide). Table 1 lists some of the known CHYSEL peptides.

TABLE 1
Viral CHYSEL peptide sequences and associated GSG-modified CHYSEL peptides.
			SEQ
	Amino acid sequence of known	Amino acid sequence of GSG-modified	ID
Organism	CHYSEL peptide	CHYSEL peptide (SEQ ID NO:)	NO:
EMC-B	GIFNAHYAGYFADLLIHDIETNPGP	GSGGIFNAHYAGYFADLLIHDIETNPGP	27
EMC-D	GIFNAHYAGYFADLLIHDIETNPGP	GSGGIFNAHYAGYFADLLIHDIETNPGP	28
EMC-PV21	RIFNAHYAGYFADLLIHDIETNPGP	GSGRIFNAHYAGYFADLLIHDIETNPGP	29
MENGO	HVFETHYAGYFSDLLIHDVETNPGP	GSGHVFETHYAGYFSDLLIHDVETNPGP	30
TME-GD7	KAVRGYHADYYKQRLIHDVEMNPGP	GSGKAVRGYHADYYKQRLIHDVEMNPGP	31
TME-DA	RAVRAYHADYYKQRLIHDVEMNPGP	GSGRAVRAYHADYYKQRLIHDVEMNPGP	32
TME-BEAN	KAVRGYHADYYRQRLIHDVETNPGP	GSGKAVRGYHADYYRQRLIHDVETNPGP	33
Theiler's-Like Virus	KHVREYHAAYYKQRLMHDVETNPGP	GSGKHVREYHAAYYKORLMHDVETNPGP	34
Ljungan virus (174F)	MHSDEMDFAGGKFLNQCGDVETNPGP	GSGMHSDEMDFAGGKFLNQCGDVETNPGP	35
Ljungan virus (145SL)	MHNDEMDYSGGKFLNQCGDVESNPGP	GSGMHNDEMDYSGGKFLNQCGDVESNPGP	36
Ljungan virus (87-012)	MHSDEMDFAGGKFLNQCGDVETNPGP	GSGMHSDEMDFAGGKFLNQCGDVETNPGP	37
Ljungan virus (M1146)	YHDKDMDYAGGKFLNQCGDVETNPGP	GSGYHDKDMDYAGGKFLNQCGDVETNPGP	38
FMD-A10	LLNFDLLKLAGDVESNPGP	GSGLLNFDLLKLAGDVESNPGP	39
FMD-A12	LLNFDLLKLAGDVESNPGP	GSGLLNFDLLKLAGDVESNPGP	40
FMD-C1	LLNFDLLKLAGDVESNPGP	GSGLLNFDLLKLAGDVESNPGP	41
FMD-O1G	LLNFDLLKLAGDMESNPGP	GSGLLNFDLLKLAGDMESNPGP	42
FMD-O1K	LTNFDLLKLAGDVESNPGP	GSGLTNFDLLKLAGDVESNPGP	43
FMD-O (Taiwan)	LLNFDLLKLAGDVESNPGP	GSGLLNFDLLKLAGDVESNPGP	44
FMD-O/SK	LLSFDLLKLAGDVESNPGP	GSGLLSFDLLKLAGDVESNPGP	45
FMD-SAT3	MCNFDLLKLAGDVESNPGP	GSGMCNFDLLKLAGDVESNPGP	46
FMD-SAT2	LLNFDLLKLAGDVESNPGP	GSGLLNFDLLKLAGDVESNPGP	47
ERAV	CTNYSLLKLAGDVESNPGP	GSGCTNYSLLKLAGDVESNPGP	48
ERBV	GATNFSLLKLAGDVELNPGP	GSGGATNFSLLKLAGDVELNPGP	49
ERV-3	GATNFDLLKLAGDVESNPGP	GSGGATNFDLLKLAGDVESNPGP	50
PTV-1	GPGATNFSLLKQAGDVEENPGP	GSGGPGATNFSLLKQAGDVEENPGP	51
PTV-2	GPGATNFSLLKQAGDVEENPGP	GSGGPGATNFSLLKQAGDVEENPGP	52
PTV-3	GPGASSFSLLKQAGDVEENPGP	GSGGPGASSFSLLKQAGDVEENPGP	53
PTV-4	GPGASNFSLLKQAGDVEENPGP	GSGGPGASNFSLLKQAGDVEENPGP	54
PTV-5	GPGAANFSLLRQAGDVEENPGP	GSGGPGAANFSLLRQAGDVEENPGP	55
PTV-6	GPGATNFSLLKQAGDVEENPGP	GSGGPGATNFSLLKQAGDVEENPGP	56
PTV-7	GPGATNFSLLKQAGDVEENPGP	GSGGPGATNFSLLKQAGDVEENPGP	57
PTV-8	GPGATNFSLLKQAGDIEENPGP	GSGGPGATNFSLLKQAGDIEENPGP	58
PTV-9	GPGATNFSLLKQAGDVEENPGP	GSGGPGATNFSLLKQAGDVEENPGP	59
PTV-10	GPGATNFSLLKQAGDVEENPGP	GSGGPGATNFSLLKQAGDVEENPGP	60
PTV-11	GPGATNFSLLKRAGDVEENPGP	GSGGPGATNFSLLKRAGDVEENPGP	61
CrPV	FLRKRTQLLMSGDVESNPGP	GSGFLRKRTQLLMSGDVESNPGP	62
DCV	EAARQMLLLLSGDVETNPGP	GSGEAARQMLLLLSGDVETNPGP	63
ABPV	GSWTDILLLLSGDVETNPGP	GSGGSWTDILLLLSGDVETNPGP	64
ABPV isolate Poland 1	GSWTDILLLLSGDVETNPGP	GSGGSWTDILLLLSGDVETNPGP	65
ABPV isolate Hungary 1	GSWTDILLLWSGDVETNPGP	GSGGSWTDILLLWSGDVETNPGP	66
IFV	TRAEIEDELIRAGIESNPGP	GSGTRAEIEDELIRAGIESNPGP	67
TaV	RAEGRGSLLTCGDVEENPGP	GSGRAEGRGSLLTCGDVEENPGP	68
EEV	QGAGRGSLVTCGDVEENPGP	GSGQGAGRGSLVTCGDVEENPGP	69
APV	NYPMPEALQKIIDLESNPPP	GSGNYPMPEALQKIIDLESNPPP	70
KBV	GTWESVLNLLAGDIELNPGP	GSGGTWESVLNLLAGDIELNPGP	71
PnPV (a)	AQGWVPDLTVDGDVESNPGP	GSGAQGWVPDLTVDGDVESNPGP	72
PnPV (b)	IGGGQKDLTQDGDIESNPGP	GSGIGGGQKDLTQDGDIESNPGP	73
Ectropis oblique picorna-	AQGWAPDLTQDGDVESNPGP	GSGAQGWAPDLTODGDVESNPGP	74
like virus (A)
Ectropis obliqua picorna-	IGGGQRDLTQDGDIESNPGP	GSGIGGGQRDLTQDGDIESNPGP	75
like virus (B)
Providence virus (a)	VGDRGSLLTCGDVESNPGP	GSGVGDRGSLLTCGDVESNPGP	76
Providence virus (b)	SGGRGSLLTAGDVEKNPGP	GSGSGGRGSLLTAGDVEKNPGP	77
Providence virus (c)	GDPIEDLTDDGDIEKNPGP	GSGGDPIEDLTDDGDIEKNPGP	78
Bovine Rotavirus	SKFQIDRILISGDIELNPGP	GSGSKFQIDRILISGDIELNPGP	79
Porcine Rotavirus	AKFQIDKILISGDVELNPGP	GSGAKFQIDKILISGDVELNPGP	80
Human Rotavirus	SKFQIDKILISGDIELNPGP	GSGSKFQIDKILISGDIELNPGP	81

As indicated above, the PQR linker encodes a “modified” CHYSEL peptide in which the tripeptide “GSG” has been added to the amino (i.e.g, NH₂) terminus of the wild-type CHYSEL peptide. In an embodiment, the PQR linker encodes any CHYSEL peptide listed in Table 1 to which the tripeptide “GSG” has been added to the amino terminus (such as those listed in the third column of Table 1).

In another embodiment, the PQR linker encodes a peptide encompassed in the consensus sequence of a CHYSEL peptide which has been modified to bear a “GSG” tripeptide at the amino (NH₂) terminus. Table 2 provides a comparison of various CHYSEL peptides as well as associated consensus sequences. For example, the PQR linker can encode a peptide encompassed in the consensus sequence from the F2A, E2A, T2A and P2A peptides (as shown in the amino acid sequence of SEQ ID NO: 21, 5^th entry on Table 2) to which as GSG tripeptide has been added (as shown in the amino acid sequence of SEQ ID NO: 23).

(SEQ ID NO: 21)
X1X2X3X4X5X6X7X8X9LX10X11X12GDVEX13NPGP
(SEQ ID NO: 23)
GSGX1X2X3X4X5X6X7X8X9LX10X11X12GDVEX13NPGP

In both the amino acid sequences of SEQ ID NO: 21 and 23, X₁ is V or absent; X₂ is K or absent; X₃ is Q or absent; X₄ is T, C, A or absent; X₅ is L, T or E; X₆ is N or G; X₇ is F, Y or R; X₈ is D, A, G or S; X₉ is L or S; X₁₀ is K or L; X₁₁ is L, T or Q; X₁₂ is A or C and X₁₃ is S or E.

TABLE 2

Comparison of CHYSEL peptides reveals a conserved sequence of amino acids (taken

from Szymczak et al., 2004). First line corresponds to the F2A peptide, second line

corresponds to the E2A peptide, third line corresponds to the T2A peptide and the

fourth line corresponds to the P2A peptide. The fifth line corresponds to the consensus

sequence obtained from comparing the F2A, E2A, T2A and P2A peptides. The sixth

line corresponds to the consensus obtained from comparing T2A and P2A peptides.

Description

−21

−20

−19

−18

−17

−16

−15

−14

−13

−12

−11

−10

−9

−8

−7

−6

−5

−4

−3

−2

−1

0

F2A (SEQ ID NO: 17)

V

K

Q

T

L

N

F

D

L

L

K

L

A

G

D

V

E

S

N

P

G

P

E2A (SEQ ID NO: 18)

—

—

Q

C

T

N

Y

A

L

L

K

L

A

G

D

V

E

S

N

P

G

P

T2A (SEQ ID NO: 19)

—

—

—

—

E

G

R

G

S

L

L

T

C

G

D

V

E

E

N

P

G

P

P2A (SEQ ID NO: 20)

—

—

—

A

T

N

F

S

L

L

K

Q

A

G

D

V

E

E

N

P

G

P

Consensus (all)

X1

X2

X3

X4

X5

X6

X7

X8

X9

L

X10

X11

X12

G

D

V

E

X13

N

P

G

P

(SEQ ID NO: 21)

Consensus T2A/P2A

—

—

—

X14

X15

X16

X17

X18

X19

L

X20

X21

X22

G

D

V

E

E

N

P

G

P

(SEQ ID NO: 22)

In another example, the PQR linker can encode a peptide encompassed in the consensus sequence from the T2A and P2A peptides (as shown in the amino acid sequence of SEQ ID NO: 22, 6^th entry on Table 2) to which as GSG tripeptide has been added (as shown in the amino acid sequence of SEQ ID NO: 24):

(SEQ ID NO: 22)
X14X15X16X17X18X19LX20X21X22GDVEENPGP
(SEQ ID NO: 24)
GSGX14X15X16X17X18X19LX20X21X22GDVEENPGP

In both the amino acid sequences of SEQ ID NO: 22 and 24, X₁₄ is A or absent; X₁₅ is E or T; X₁₆ is G or N; X₁₇ is R or F; X₁₈ is G or S; X₁₉ is S or L; X₂₀ is L or K; X₂₁ is T or Q and X₂₂ is C or A.

In some embodiments, the PQR can include one or more additional amino acids of the CHYSEL peptide which is/are found upstream (from the N-terminus) of those presented in Table 2. For example, the PQR can include up to 11 additional amino acids found upstream of the amino acid sequences shown in Table 2. If the PQR includes one or more of upstream amino acid, it also should include a “GSG” tri-peptide linker at its N-terminal end. Exemplary longer CHYSEL peptides are can have the amino acid sequence of SEQ ID NO: 96 to which a “GSG” tri-peptide linker has been added at its N-terminal end.

In yet a further embodiment, the PQR linker can encode the P2A peptide from the porcine teschovirus-1 (SEQ ID NO: 1) modified to bear the GSG tripeptide (SEQ ID NO: 3). In still a further embodiment, the PQR linker can encode the T2A peptide from the Thosea asigna insect virus (SEQ ID NO: 2) modified to bear the GSG tripeptide (SEQ ID NO: 26).

In the context of the present disclosure, the PQR linker is a nucleic acid molecule which encodes the cleavable peptide disclosed herein and is capable of being transcribed into a messenger RNA molecule. The PQR linker molecules can have the generic nucleic acid sequence set forth in SEQ ID NO: 25:

(SEQ ID NO: 25)
N1N2N3 N4N5N6 N7N8N9 N10N11N12 N13N14N15 N16N17
N18 N19N20N21 N22N23N24 N25N26N27N28N29N30 N31
N32N33 N34N35N36 N37N38N39 N40N41N42 N43N44N45 N46
N47N48 N49N50N51N52N53N54N55N56N57 N58N59
N60 N61N62N63 AAY CCN64 GGA CCN65

In the nucleic acid sequence of SEQ ID: 25, N₁ to N₆₃ represent any nucleic acid capable of forming codons encoding the subsequence GSGX₁X₂X₃X₄X₅X₆X₇X₈X₉LX₁₀X₁₁X₁₂GDVEX₁₃ of SEQ ID NO: 23 (corresponding to residues 1 to 21 of SEQ ID NO: 23) or the subsequence GSGX₁₄X₁₅X₁₆X₁₇X₁₈X₁₉LX₂₀X₂₁X₂₂GDVEE of SEQ ID NO: 24 (corresponding to residues 1 to 18 of SEQ ID NO: 24). Further, N₆₄ can be T or U and N₆₅ can be T or U.

When the PQR linker is a DNA molecule, it can be represented by the nucleic acid sequence of SEQ ID NO: 26:

(SEQ ID NO: 26)
N1N2N3 N4N5N6 N7N8N9 N10N11N12 N13N14N15 N16N17
N18 N19N20N21 N22N23N24 N25N26N27N28N29N30 N31
N32N33 N34N35N36 N37N38N39 N40N41N42 N43N44N45 N46
N47N48 N49N50N51 N52N53N54N55N56N57 N58N59
N60 N61N62N63 AAY CCT GGA CCT

In the nucleic acid sequence of SEQ ID NO: 26, N₁ to N₆₃ represent any nucleic acid capable of forming codons encoding the subsequence GSGX₁X₂X₃X₄X₅X₆X₇X₈X₉LX₁₀X₁₁X₁₂GDVEX₁₃ of SEQ ID NO: 23 (corresponding to residues 1 to 21 of SEQ ID NO: 23) or the subsequence GSGX₁₄X₁₅X₁₆X₁₇X₁₈X₁₉LX₂₀X₂₁X₂₂GDVEE of SEQ ID NO: 24 (corresponding to residues 1 to 18 of SEQ ID NO: 24).

Exemplary nucleic acid sequences capable of encoding the cleavable peptides include those presented at SEQ ID NO: 4 (wild-type sequence from the porcine teschovirus) as well as at SEQ ID NO: 6 (wild-type sequence from the thosea asigna insect virus).

It is important that the codons of the nucleic acid sequence of the PQR linker encoding the subsequence NPGP of SEQ ID NO: 23 (corresponding to residues 22 to 25 of SEQ ID NO: 23) or 24 (corresponding to residues 19 to 22 of SEQ ID NO: 24) be identical to the codons used in the wild-type 2A and 2A-like peptides. As such, the codons of the PQR linker encoding the subsequence NPGP of SEQ ID NO: 23 (corresponding to residues 22 to 25 of SEQ ID NO: 23) or 24 (corresponding to residues 19 to 22 of SEQ ID NO: 24) correspond to:

AAY CCN₆₄ GGA CCN₆₅ (residues 64 to 75 of SEQ ID NO: 25, in which N₆₄ and N₆₅ can independently be T or U), when the PQR linker is a DNA or a RNA molecule; or

AAY CCT GGA CCT (residues 55 to 66 of SEQ ID NO: 26), when the PQR linker is a DNA molecule.

In an embodiment, the PQR linker is a ribonucleic acid (RNA) molecule. In another embodiment, the PQR linker is a deoxyribonucleic acid (DNA) molecule. In yet another embodiment, the PQR linker can be a nucleic acid molecule including both ribonucleic acid nucleotides and deoxyribonucleic nucleotides (i.e., a DNA/RNA mixture).

The codons encoding the subsequence X₁X₂X₃X₄X₅X₆X₇X₈X₉LX₁₀X₁₁X₁₂GDVEX₁₃ of SEQ ID NO: 23 (corresponding to residues 4 to 21 of SEQ ID NO: 23) or the subsequence X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉LX₂₀X₂₁X₂₂GDVEE of SEQ ID NO: 24 (corresponding to residues 4 to 18 of SEQ ID NO: 24) can be modified to increase the cleavage of the peptide and/or the stoichiometric ratio between the reporter protein and the protein of interest. For example, one or more codons encoding X₁X₂X₃X₄X₅X₆X₇X₈X₉LX₁₀X₁₁X₁₂GDVEX₁₃ of SEQ ID NO: 23 (corresponding to residues 4 to 21 of SEQ ID NO: 23) or the subsequence X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉LX₂₀X₂₁X₂₂GDVEE of SEQ ID NO: 24 (corresponding to residues 4 to 18 of SEQ ID NO: 24) can be selected to correspond to the least preferred codon used in a particular host.

It is known in the art that various codons can encode the same amino acid and that different organism use some codons preferentially. It has been surprisingly found herein that when the codons encoding the subsequence X₁X₂X₃X₄X₅X₆X₇X₈X₉LX₁₀X₁₁X₁₂GDVEX₁₃ of SEQ ID NO: 23 (corresponding to residues 4 to 21 of SEQ ID NO: 23) or the subsequence X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉LX₂₀X₂₁X₂₂GDVEE of SEQ ID NO: 24 (corresponding to residues 4 to 18 of SEQ ID NO: 24) were selected to include the most preferred codons used in a particular host, the cleavage of the PQR linker in the particular host was substantially decreased, which prevented quantifying the protein of interest. On the other hand, when the codons encoding the subsequence X₁X₂X₃X₄X₅X₆X₇X₈X₉LX₁₀X₁₁X₁₂GDVEX₁₃ of SEQ ID NO: 23 (corresponding to residues 4 to 21 of SEQ ID NO: 23) or the subsequence X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉LX₂₀X₂₁X₂₂GDVEE of SEQ ID NO: 24 (corresponding to residues 4 to 18 of SEQ ID NO: 24) were selected to include some least preferred codons used in a particular host (e.g., de-optimized), the cleavage of the PQR linker was increased in the particular host, which, in some embodiments, allowed protein quantification.

Consequently, in an embodiment, at least some of the codons encoding the subsequence X₁X₂X₃X₄X₅X₆X₇X₈X₉LX₁₀X₁₁X₁₂GDVEX₁₃ of SEQ ID NO: 23 (corresponding to residues 4 to 21 of SEQ ID NO: 23) or the subsequence X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉LX₂₀X₂₁X₂₂GDVEE of SEQ ID NO: 24 (corresponding to residues 4 to 18 of SEQ ID NO: 24) are not those which are preferably used in the host in which the protein quantification is intended to be performed. In still another embodiment, at least one of the codons encoding the subsequence X₁X₂X₃X₄X₅X₆X₇X₈X₉LX₁₀X₁₁X₁₂GDVEX₁₃ of SEQ ID NO: 23 (corresponding to residues 4 to 21 of SEQ ID NO: 23) or the subsequence X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉LX₂₀X₂₁X₂₂GDVEE of SEQ ID NO: 24 (corresponding to residues 4 to 18 of SEQ ID NO: 24) is selected to be the least preferred codon used in the host in which the protein quantification is intended to be performed. In yet another embodiment, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 of the codons encoding the subsequence X₁X₂X₃X₄X₅X₆X₇X₈X₉LX₁₀X₁₁X₁₂GDVEX₁₃ of SEQ ID NO: 23 (corresponding to residues 4 to 21 of SEQ ID NO: 23) or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 of the codons encoding the subsequence X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉LX₂₀X₂₁X₂₂GDVEE of SEQ ID NO: 24 (corresponding to residues 4 to 18 of SEQ ID NO: 24) are selected to be the least preferred codon used in the host in which the protein quantification is intended to be performed. In still a further embodiment, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of the codons encoding the subsequence X₁X₂X₃X₄X₅X₆X₇X₈X₉LX₁₀X₁₁X₁₂GDVEX₁₃ of SEQ ID NO: 23 (corresponding to residues 4 to 21 of SEQ ID NO: 23) or the subsequence X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉LX₂₀X₂₁X₂₂GDVEE of SEQ ID NO: 24 (corresponding to residues 4 to 18 of SEQ ID NO: 24) are selected to be the least preferred codon used in the host in which the protein quantification is intended to be performed. In yet another embodiment, all the codons encoding the subsequence X₁X₂X₃X₄X₅X₆X₇X₈X₉LX₁₀X₁₁X₁₂GDVEX₁₃ of SEQ ID NO: 23 (corresponding to residues 4 to 21 of SEQ ID NO: 23) or the subsequence X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉LX₂₀X₂₁X₂₂GDVEE of SEQ ID NO: 24 (corresponding to residues 4 to 18 of SEQ ID NO: 24) are selected to be the least preferred codon used in the host in which the protein quantification is intended to be performed. In embodiments in which some but not all codons encoding the subsequence X₁X₂X₃X₄X₅X₆X₇X₈X₉LX₁₀X₁₁X₁₂GDVEX₁₃ of SEQ ID NO: 23 (corresponding to residues 4 to 21 of SEQ ID NO: 23) or the subsequence X₁₄X₁₅X₁₆X₁₇X₁₈X₁₉LX₂₀X₂₁X₂₂GDVEE of SEQ ID NO: 24 (corresponding to residues 4 to 18 of SEQ ID NO: 24), the least preferred codons used can be located preferentially at the 5′ terminus of the nucleic acid molecule.

In embodiments in which the PQR linker encodes additional amino acids than those presented in SEQ ID NO: 23 or SEQ ID NO: 24, the PQR linker can include, for those additional amino acid, one or more least-favored codons being used.

To select codons least preferred in a particular host, it is possible to refer to Tables 3A and 3B below. Such tables include the most (Table 3A) and the least preferred (Table 3B) codons in function of the host. In order to include codons least preferred from a particular host, it is possible to select the codon associate to the particular host in Table 3B and/or to exclude the most preferred codon associated to the particular host in Table 3A.

Exemplary nucleic acid sequences containing codons which are least preferred in mammalian or Drosophila hosts include, but are not limited to the nucleic acid molecule having the nucleic acid sequence shown in SEQ ID NO: 9, 10, 11, 12, 14, 15 or 16.

TABLE 3A
Most preferred codons used for amino acids in function of origin
of the host.
				Yeast	Insect	Bacteria
Amino Acid	Human	Mouse	Rat	(S. cerevisea)	(D. melanogaster)	(E. coli)
Alanine (A)	GCC	GCC	GCC	GCT	GCC	GCC
Arginine (R)	CGG	CGG	AGG	AGA	CGC	CGC
Asparagine (N)	AAC	AAC	AAC	AAT	AAC	AAC
Aspartic acid	GAC	GAC	GAC	GAT	GAT	GAT
(D)
Cysteine (C)	TGC	TGC	TGC	TGT	TGC	TGC
Glutamine (Q)	CAG	CAG	CAG	CAA	CAG	CAG
Glutamic acid	GAG	GAG	GAG	GAA	GAG	GAA
(E)
Glycine (G)	GGC	GGC	GGC	GGT	GGC	GGC
Histidine (H)	CAC	CAC	CAC	CAT	CAC	CAT
Isoleucine (I)	ATC	ATC	ATC	ATT	ATC	ATT
Leucine (L)	CTG	CTG	CTG	TTG	CTG	CTG
Lysine (K)	AAG	AAG	AAG	AAA	AAG	AAA
Methionine (M)	ATG	ATG	ATG	ATG	ATG	ATG
Phenylalanine	TTC	TTC	TTC	TTT	TTC	TTT
(F)
Proline (P)	CCC	CCC	CCC	CCA	CCC	CCG
Serine (S)	AGC	AGC	AGC	TCT	AGC	AGC
Threonine (T)	ACC	ACC	ACC	ACT	ACC	ACC
Tryptophan (W)	TGG	TGG	TGG	TGG	TGG	TGG
Tyrosine (Y)	TAC	TAC	TAC	TAT	TAC	TAT
Valine (V)	GTG	GTG	GTG	GTT	GTG	GTG

TABLE 3B
Least preferred codon used for amino acids in function of origin
of the host. (—) indicates that, besides the codon listed in Table 3A,
no other codon exists for this amino acid.
				Yeast	Insect	Bacteria
Amino Acid	Human	Mouse	Rat	(S. cerevisea)	(D. melanogaster)	(E. coli)
Alanine (A)	GCG	GCG	GCG	GCG	GCA	GCT
Arginine (R)	CGT	CGT	CGT	CGG	CGG	CGA
Asparagine (N)	AAT	AAT	AAT	AAC	AAT	AAT
Aspartic acid (D)	GAT	GAT	GAT	GAC	GAC	GAC
Cysteine (C)	TGT	TGT	TGT	TGC	TGT	TGT
Glutamine (Q)	CAA	CAA	CAA	CAG	CAA	CAA
Glutamic acid	GAA	GAA	GAA	GAG	GAA	GAG
(E)
Glycine (G)	GGT	GGT	GGT	GGG	GGG	GGA
Histidine (H)	CAT	CAT	CAT	CAC	CAT	CAC
Isoleucine (I)	ATA	ATA	ATA	ATC	ATA	ATA
Leucine (L)	CTA	CTA	CTA	CTC	CTA	CTA
Lysine (K)	AAA	AAA	AAA	AAG	AAA	AAG
Methionine (M)	—	—	—	—	—	—
Phenylalanine	TTT	TTT	TTT	TTC	TTT	TTC
(F)
Proline (P)	CCG	CCG	CCG	CCG	CCT	CCC
Serine (S)	TCG	TCG	TCG	TCG	TCT	TCG
Threonine (T)	ACG	ACG	ACG	ACG	CCT	ACA
Tryptophan (W)	—	—	—	—	—	—
Tyrosine (Y)	TAT	TAT	TAT	TAC	TAT	TAC
Valine (V)	GTA	GTA	GTA	GTG	GTA	GTA

Vector Comprising the PQR Linker and Associated Tools

The PQR linker can be presented in the form of a vector which is at least designed to also encode a reporter protein and a protein of interest. In the methods described herein, the PQR linker is intended to be located between the two proteins, i.e., between a nucleic acid sequence encoding a protein of interest and a nucleic acid sequence encoding the reporter protein. In the context of the present disclosure, the nucleic acid molecule of the PQR linker is referred to as the “first” nucleic acid molecule, the nucleic acid molecule encoding the reporter protein is referred as the “second” nucleic acid molecule and the nucleic acid molecule encoding the protein of interest is referred to as the “third” nucleic acid molecule.

In its simplest embodiment, the vector comprises the first nucleic acid molecule (i.e., the PQR linker) and is designed to allow for the subsequent integration of the second nucleic acid molecule (i.e., encoding the reporter protein) and of the third nucleic acid molecule (i.e., encoding the protein of interest) on each side of the first nucleic acid molecule. The vector must be designed to allow for the transcription of a mRNA encoding the entire poly-protein sequence, comprising the PQR linker flanked on each side by the reporter protein and the protein of interest. This embodiment of the vector allows a maximum of flexibility for the end-user to select a particular reporter protein and a particular protein of interest. In an embodiment, the second nucleic acid molecule (i.e., encoding the reporter protein) is intended to be located upstream of the first nucleic acid molecule and the third nucleic acid molecule (i.e., encoding the protein of interest) is intended to be located downstream of the first nucleic acid molecule. Alternatively, the second nucleic acid molecule (i.e., encoding the reporter protein) can be intended to be located downstream of the first nucleic acid molecule while the third nucleic acid molecule (i.e., encoding the protein of interest) can be intended to be located upstream of the first nucleic acid molecule.

In another embodiment, the vector can comprise both the PQR linker and the second nucleic acid sequence (i.e., encoding the reporter protein). In this embodiment, the end-user is provided with a customizable vector in which the third nucleic acid molecule (i.e., encoding the protein of interest) can be inserted and used. In this embodiment, the second nucleic acid molecule (i.e., encoding the reporter protein) can be located upstream of the first nucleic acid molecule and the third nucleic acid molecule (i.e., encoding the protein of interest) is intended to be located downstream of the first nucleic acid molecule. Alternatively, the second nucleic acid molecule (i.e., encoding the reporter protein) can be located downstream of the first nucleic acid molecule and the third nucleic acid molecule (i.e., encoding the protein of interest) is intended to be located upstream of the first nucleic acid molecule.

In another embodiment, the vector can comprise both the PQR linker and the third nucleic acid sequence (i.e., encoding the protein of interest). In this embodiment, the end-user is provided with a customizable vector in which the second nucleic acid molecule (i.e., encoding the reporter protein) can be inserted and used. In this embodiment, the second nucleic acid molecule (i.e., encoding the reporter protein) is intended to be located upstream of the first nucleic acid molecule and the third nucleic acid molecule (i.e., encoding the protein of interest) can be intended to be located downstream of the first nucleic acid molecule. Alternatively, the second nucleic acid molecule (i.e., encoding the reporter protein) is intended to be located downstream of the first nucleic acid molecule and the third nucleic acid molecule (i.e., encoding the protein of interest) can be intended to be located upstream of the first nucleic acid molecule.

In yet another embodiment, the vector can comprise the PQR linker, the second nucleic acid sequence (i.e., encoding the reporter protein) and the third nucleic acid molecule (i.e., encoding the protein of interest). In this embodiment, the end-user is provided with a ready-to-use vector to quantify a specific protein of interest using a specific reporter protein. In this embodiment, the second nucleic acid molecule (i.e., encoding the reporter protein) can be located upstream of the first nucleic acid molecule and the third nucleic acid molecule (i.e., encoding the protein of interest) can be located downstream of the first nucleic acid molecule. Alternatively, the second nucleic acid molecule (i.e., encoding the reporter protein) can be located downstream of the first nucleic acid molecule and the third nucleic acid molecule (i.e., encoding the protein of interest) can be located upstream of the first nucleic acid molecule.

The vectors described herein are designed to allow for the expression of one or more fusion protein (comprising the reporter protein and the protein of interest as well as the PQR linker). When a plurality of proteins of interest or reporter proteins are transcribed from the vector, each protein can be the same or different and comprises a reporter protein, a protein of the interest and a PQR linker between the reporter protein and the protein of interest.

In a further embodiment, the vector can be a linear vector or a circular vector. The vector can also be an integratable vector and as such can comprise a nucleic acid sequence capable of favoring or allowing integration of the vector in the genome of the host cell. In such embodiment, once integrated, some of the sequence of the original vector may have been removed during integration. In another embodiment, the vector can replicate independently from the host genome and as such can comprise a suitable origin of replication. The vector can also include a further nucleic acid molecule encoding a selection marker protein to identify host cells bearing the vector from those not bearing the vector.

In yet another embodiment, the vector can further comprise, upstream of the sequence encoding the poly-protein a regulatory sequence (promoter, enhancer and the like) for allowing the transcription of the mRNA of the poly-protein. If it is intended to study the expression of the protein of interest in its wild-type environment, it is possible to use the regulatory region associated with the protein of interest upstream of the poly-protein. It is also possible to introduce such vector in a host cell which has been previously knocked-down or knocked out for expression of the protein of interest. In other embodiments, it is possible to use other regulatory regions (e.g., constitutive or inducible regulatory regions) upstream of the poly-protein.

In still another embodiment, the vector can be designed to be integrated in the host's genome either in an unspecific or a specific manner (e.g., using the CRISPR/Cas9 system).

In the vectors described herein, the second nucleic acid molecule encodes a reporter protein. In the context of the present disclosure, a reporter protein is a protein which generates a quantifiable signal (either endogenously or via its enzymatic or biologic activity). The reporter protein can be, for example a fluorescent protein (such as, for example, a green fluorescent protein (GFP), a red fluorescent protein (RFP), a yellow fluorescent protein (YFP), a blue fluorescent protein (BFP) or a cyan fluorescent protein (CFP)), an antibiotic-resistance protein, an immunoglobulin or a immunoglobulin fragment, an ion channel, a transcription factor, a ribosomal protein, an enzyme and/or a receptor.

The vector can be any vector suitable for expressing the mRNA encoding the poly-protein. For example, the vector can be derived from a virus (e.g., retrovirus, adenovirus, herpes or vaccinia), from a yeast (e.g., an artificial chromosome or cosmid), from a bacteria (e.g., a bacterial plasmid for example), or from a wholly synthetic sequence.

The present disclosure can also provide a control vector which encodes for a control poly-protein which comprises a reporter protein, a protein of interest and a control PQR linker between the reporter protein and the protein of interest. The control PQR linker is not cleaved during the translation of the mRNA encoding the control fusion protein. The control vector can be used as a negative control to determine the signal associated with the reporter protein in an uncleaved form (i.e., while remaining in the fusion protein). As such, the vector and the control vector can be used together and should preferably comprise the same reporter protein, the same protein of interest, but a different PQR (one that can be cleaved in the vector, and one that cannot be cleaved in the control vector).

The present disclosure also provides for a kit for performing protein quantification using the vector described herein. In its simplest embodiment, the kit comprises the vector described herein and instructions on how to use the vector to quantify the protein of interest. For example, the instructions can indicate how to introduce the second nucleic acid molecule in the vector, how to introduce the third nucleic acid molecule in the vector, how to introduce the vector in a host cell, how to integrate the vector into the genome, how to select for host cell bearing the vector and/or how to measure the signal from the cleaved reporter protein. The kit can also provide a control vector and instructions on how to use the control vector to quantify the protein of interest. The kit can further provide a host cell and instructions on how to use the control vector to quantify the protein of interest.

The present disclosure also provides for a host cell or host organism comprising the nucleic acid molecule encoding the protein of interest, PQR linker and reporter (either in the form of a vector (independent or integrated in the genome) or in the form of an integrated nucleic acid molecule. The host cell, or host cell in a multi-cellular host organism, is capable of transcribing the nucleic acid molecule encoding the protein of interest and including the PQR linker and reporter. The host cell can be any eukaryotic cell. Exemplary eukaryotic cells include mammalian cells (such as human cells, rodent cells), other animal cells (such as fish cells, amphibian cells, insect cells, worm cells), plant cells, algal cells, fungal cells (such as yeast cells and mold cells).

Method for Quantifying Proteins in Host Cells

The present disclosure also provides method for quantifying a protein of interest in a host cell. The protein can be measured in vitro (when the host cell can be maintained in in vitro conditions), in vivo (when the host cell is located in a multicellular organism) or ex vivo (when the host cell is removed from a multicellular organism). In some embodiments, the protein can even be measured in living cells. In some specific embodiments, the protein can be measure at the single-cell level.

In the context of the present disclosure, the nucleic acid transcript associated with the poly-protein is a single nucleic acid molecule which is capable of being cleaved inside the PQR linker (between the codon encoding the penultimate glycine residue and the codon encoding the ultimate proline residue of the PQR linker). As such, the translation of the nucleic acid transcript of the poly-protein can generate two distinct proteins: the reporter protein and the protein of interest. Depending on which side the two proteins are located, these proteins will also contain one or more residues of the PQR linker. The protein located upstream of the PQR linker in the nucleic acid transcript will bear, at its carboxy (COOH) terminus all the residues of the PQR linker, except the ultimate proline residue. The protein located downstream of the PQR linker in the nucleic acid transcript will bear, at its amino (NH²) terminus, the ultimate proline residue of the PQR linker.

The first step of this method requires that the vector encoding the poly-protein (which includes the PQR linker) be expressed in a host cell. Expression of the poly-protein can be driven from regulatory sequences present in the vector, upstream or downstream, of the poly-protein. Alternatively, expression of the poly-protein can be driven from endogenous regulatory sequences present in the host's genome by integrating the poly-protein specifically in the host's genome. The method can be practiced on any eukaryotic host cell which can transcribe the poly-protein in a poly-cistronic nucleic acid transcript and translate the resulting nucleic acid transcript. Without limitation the host cell can be a mammalian (such as a human), a plant, an insect, a yeast, a mold, and/or an algae.

The method can be designed to accommodate the quantification of more than one protein of interest. In order to do so, more than one PQR and reporter protein are encoded on the same vector or more than one vector is transferred inside the host cell. Preferably, each poly-protein comprises a distinct protein of interest and a distinct reporter protein. Care should also be taken when combining two or more reporter proteins in the same cell so as to avoid or minimize an overlap in the signal associated with each of the reporter proteins.

This first step may optionally include constructing the vector to include a reporter protein and/or a protein of interest, transferring the vector inside the host cell, integrating the vector inside the genome of the host cell and/or manipulating (for example, knocking down) the endogenous expression of the protein in the host cell. As indicated above, the nucleic acid sequence of the PQR linker is de-optimized (i.e., modified to include the least favored codons) in function of the host cell on which the quantification method will be practiced.

Once the nucleic acid transcript associated with the poly-protein is expressed, it can be cleaved during the translation process to generate, at a stoichiometric ratio (and in some embodiment, in an equimolar ratio), a cleaved reporter protein and a cleaved protein of interest. The next step of the method is thus to measure the signal associated with the cleaved reporter protein to estimate or quantify the amount of the protein of interest. The measure of the signal can be repeated in time or conducted only once.

The second step is dependent on the type of reporter protein being used. For example, if the reporter protein is a fluorescent protein, then this second step will include a determination of fluorescence. In such an example, it may be necessary determine the fluorescence which is specific to the cleaved reporter protein and/or to determine the background fluorescence which is not associated with the cleaved reporter protein. In another example, when the reporter protein is an enzyme, the second step can include contacting the enzyme with a substrate which will, upon the enzyme's activity, provide or remove a signal which can be measured and, optionally be quantified. In such an example, it may be necessary to provide a control value in the absence of the substrate. In still another example, if the reporter protein is an antibody, then this second step could include a determination of the antibody amount (either by flow cytometry, an ELISA and the like). In yet another example, if the reporter protein is an ion channel, then this second step could include measuring the activity associated by the channel.

Once the signal associated with the reporter protein has been obtained, it is used to estimate the amount of the protein of interest. For example, the signal can be graphically compared to a standard curve associating the fluorescence to the amount of the protein of interest. In another example, the PQR fluorescence signal of a protein of interest can be compared to the fluorescence signal (i.e., in another channel) of another protein of interest, for normalization or for analysis of differential protein production. In another example, and as described herein, the estimation of the protein based on the signal of the reporter protein with can be done through a linear regression technique. A linear regression that goes through the origin (0,0) could be performed between a standard (offline) measure of the protein of interest and the signal of the reporter protein. The slope of this regression (and the y intercept) enables the conversion of the fluorescent signal to the estimated value for the parameter. In another example, the PQR fluorescence signal of a protein of interest can be measured and compared against a measured phenotype, in a single cell. This can be used to determine the relationship between protein concentration and cellular phenotype. In a further example, the PQR fluorescence signal can be measured over time in the same cell to quantify the change in protein production over time, such as before and after an experimental manipulation, drug induction, or intervention.

As it will be shown below, a method to quantitate protein concentrations in single living cells using a fluorescent reporter was developed (FIG. 1). Modified virus sequences that allow for an equimolar separation of an upstream protein of interest and a downstream protein of interest were used, all contained within a single strand of RNA. When a fluorescent reporter is separated from the protein of interest, the number of fluorescent molecules produced are stoichiometric with the number of molecules of the protein of interest produced, and thus the fluorescence output can be used as a readout for the number of molecules of interest produced, i.e., its relative protein concentration (FIG. 1).

RNA sequences encoding peptides called cis-acting hydrolase elements (CHYSELs) can interact with the ribosome during protein translation to produce non-canonical protein coding events and separate a nascent polypeptide chain from an actively translating sequence. CHYSEL polypeptides (also known as “2A” and “2A-like” peptides, collectively) are used by RNA viruses to separate each of the viral genes to be translated. This allows for multiple proteins to be produced from the virus's single, polycistronic RNA strand. The mechanism by which separation of an upstream and downstream gene occurs is due to the specific and conserved sequence of CHYSEL residues upstream of a glycine proline separation point (FIG. 1A). In normal translation the peptidyl transferase activity of the ribosome produces the peptide bond of the growing peptide chain. The ribosome translocates and moves on to the next tRNA as the peptide chain is elongating through the exit tunnel. In the presence of the conserved CHYSEL residues that lie at the base of the exit tunnel, this forms a turn in the peptide chain that shifts the ester link between the peptide and the tRNA glycine away from the prolyl tRNA. This torsion causes the ribosome to stall and inhibits the peptidyl transferase activity, forcing the peptide chain to be released. The ribosome skips the glycyl-prolyl peptide bond, reinitiates from the proline, and translation continues with the downstream protein. This unique mechanism has been shown to produce equimolar amounts of upstream and downstream proteins, but previous high-throughput methods to quantitate bicistronic protein production have used CHYSEL peptides that do not consistently separate, and these poly-protein products contaminate the quantitation of protein concentration because they are by definition equimolar. Although CHYSEL sequences have been used for more than a decade, CHYSEL sequences were created in a novel concept to exploit the linear relationships between fluorescent molecule concentration and its fluorescence output, and fluorescent molecule concentration and the protein of interest concentration (FIG. 1B).

To create a protein quantitation reporter, CHYSEL sequences must meet two important criteria: first, separation of the protein of interest and the reporter must be close to 100% reliable, otherwise, the resulting poly-product may interfere with protein function, and second, production of the fluorescent reporter must be stoichiometric with the protein of interest, since many CHYSELs produce inconsistent stoichiometric separations depending on cell state, cell type, or at random. The production need not be equimolar at steady state levels, but consistently stoichiometric across cell states and types (FIG. 1B).

The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

Example I

Experimental Procedures

Protein Quantitation Reporter Constructs. Sequences for CHYSEL peptides were tested from Group IV, positive-sense ssRNA viruses, including the Picornaviridae family for 2A peptides, or the Permutotetraviridae family for 2A-like peptides (Diao and White, 2012; Kim et al., 2011). For our initial screens, we mostly focused on four broad CHYSEL peptide sequences from the following viruses: Equine rhinitis A virus (E2A), Foot-and-mouth disease virus (F2A), Porcine teschovirus-1 (P2A), and Thosea asigna virus (T2A) and tested for stoichiometric production and separation of fluorescent proteins and Shaker potassium channel. We added glycine and serine linkers to the N-terminus of all CHYSEL sequences tested to enhance peptide separation (Yang et al., 2008). We selected the amino acid sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 1) from the porcine teschovirus-1 for use in mammalian cells (Kim et al., 2011), and EGRGSLLTCGDVEENPGP (SEQ ID NO: 2) from the Thosea asigna insect virus for use in Drosophila cells (Diao and White, 2012). We compared codon optimization of the CHYSEL peptides versus the viral sequences of the CHYSEL peptides, and found that both the original viral sequence and the codon optimized forms resulted in a large fraction of un-separated, fusion product (FIGS. 2 and 11). Codon optimization often created a larger proportion of un-separated product, indicating that codon optimization could be worse for protein quantitation. Thus, we surmised that codon optimization could speed up ribosomal activity causing it to ignore the separation event between the final glycine and proline of the CHYSEL peptide. We tested DNA sequences that were selected for non-favored codons to decrease translation speed, which we found to enhance reliable separation (FIGS. 2 and 11) (Novoa and Ribas de Pouplana, 2012; Zhou et al., 2011). The DNA sequence chosen for the PQR in mammalian cells (P2A-derived with glycine and serine linker, codon variation 3) was: GGAAGCGGAGCGACGAATTTTAGTCTACTGAAACAAGCGGGAGACGTGGAGGAAAACC CTGGACCT (SEQ ID NO: 83). The DNA sequence chosen for the PQR in Drosophila cells (T2A-derived with glycine and serine linker, codon variation 2) was: GGAAGCGGAGAAGGTCGTGGTAGTCTACTAACGTGTGGTGACGTCGAGGAAAATCCTG GACCT (SEQ ID NO: 84). We also tested whether extended CHYSEL sequences, 30 amino acids in total length from the separation point, might enhance separation by further interacting with the exit tunnel (Luke et al., 2008). We found that these extended viral CHYSEL sequences still created a proportion of fusion product compared with shorter, 19 amino acid codon de-optimized CHYSEL sequences (FIG. 11c). Mutated PQR sequences that failed to separate were used as linkers for fusion protein experiments. All viral sequences were generated using gene synthesis into a pUC57 vector (BioBasic, Markham, ON), and cloned into pCAG for mammalian experiments or pJFRC7 for Drosophila melanogaster experiments. GFP, RFP, and BFP constructs were based on superfolderGFP, TagRFP-T, and mTagBFP2, respectively. SuperfolderGFP and TagRFP-T were chosen for their relatively fast maturation times, 6 min and 100 min, and average turnover rates at 26 hours, respectively (Corish and Tyler-Smith, 1999; Khmelinskii et al., 2012; Pedelacq et al., 2006; Shaner et al., 2008). For GFP and RFP protein concentration and fluorescence intensity measurements, proteins were purified from E. coli using GFP-specific chromatography columns (Biorad, Hercules, Calif.), and protein concentrations were measured using a Bradford assay with a NanoDrop 2000 (Thermo Fisher). Samples were serially diluted and thin samples were imaged on glass slides to reduce any non-linear effects using a standard fluorescence microscope (see Image Acquisition). ShakerGFP cDNA and hs-PER::YFP were kind gifts, and all other plasmids were obtained through Addgene (Cambridge, Mass.). GFP::RFP fusion proteins were verified using immunoblotting (FIG. 2), and imaging experiments verified that these large proteins were excluded from the nucleus.

Cell Culture. HEK293, Neuroblastoma-2A (N2A), and 22c10 cells were cultured at 37° C. under 5% CO₂ in Dulbecco's Modified Eagle Medium (Wisent, St-Bruno, QC) and H-Cell (22c10) (Wisent, St-Bruno, QC), or for Drosophila S2 and Kc cells, at 25° C. in Ex-Cell 420 Medium (Sigma-Aldrich, St. Louis, Mo.). Media for mammalian cells were supplemented with 10% fetal bovine serum (FBS) (Wisent), and 100 units/mL penicillin (Life Technologies, Carlsbad, Calif.) and 100 μg/mL streptomycin (Life Technologies). Cells were transfected with 5 pg of plasmid DNA in 35 mm dishes using Lipofectamine 3000 (Life Technologies). For genome editing experiments, 800 ng of CRISPR-Cas9 plasmid DNA were co-transfected with 800 ng of repair template circular plasmid in 12-well plates. After 2-7 days, cells were non-enzymatically dissociated and seeded on glass coverslips and prepared for imaging and electrophysiology experiments.

Immunoblotting. Immunoblot experiments were performed four times. One billion cells were placed into lysis buffer (25 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1% Triton-X) with SIGMAFAST™ protease inhibitor tablet solution (Sigma-Aldrich). Protein concentrations were measured using a bicinchoninic acid protein assay (Pierce, Rockford, Ill.) and 30-40 μg of protein was loaded into a NuPAGE Novex 12% Bis-Tris Gel (Life Technologies). Proteins were separated by electrophoresis and transferred to a polyvinylidene fluoride membrane using Invitrogen iBlot dry transfer (Life Technologies). The membrane was blocked in 5% BSA in PBS-T and incubated with the following antibody dilutions: 1:1000 anti-RFP rabbit polyclonal (R10367, Life Technologies), 1:2000 anti-GFP rabbit polyclonal (A6455, Life Technologies), and 1:5000 anti-actin JLA-20 mouse monoclonal (Developmental Studies Hybridoma Bank, Iowa City, Iowa) for the top, middle, and bottom blots, respectively, in FIGS. 2 and 11. Secondary antibodies used were 1:10 000 HRP-conjugated Donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc.) and HRP-conjugated goat anti-mouse IgG (Abcam, USA). All antibodies were dissolved in 5% BSA in PBS-T. Membranes were imaged using the Pierce ECL Chemiluminescence Detection Kit for HRP (Thermo Scientific, USA). The ratio of band intensity of GFP or fusion products was normalized to actin and quantified using ImageJ, as described (Cvetkovska et al., 2013). We performed Western blots on all PQR constructs used in experiments and confirmed the absence of fusion protein products for GFP, RFP, ShakerGFP, PER, Cut, and RPL13A proteins (FIGS. 11 and 12).

Image Acquisition. Fluorescence and bright-field microscopy was performed using a Zeiss AxioScope A1, an Olympus laser scanning confocal microscope FV1000, and a Perkin Elmer UltraView spinning disk confocal Leica DMLFSA microscope. All images were acquired at 512×512 pixels using a 40× water objective, N.A. 1.0 (epifluorescence) or 60× oil, N.A. 1.4, or 63× water, N.A. 0.9, objectives (confocal) corresponding to an 215×160 μm or 120×110 μm field of view, respectively. Fluorescence emission was detected using a charge-coupled device camera (MRm) for the Zeiss and (OrcaER, Hamamatsu) Leica microscopes, and photomultiplier tubes for the Olympus microscope. All image acquisition parameters were fixed for each imaging channel for exposure time, excitation intensity, gain, and voltages. Cells that were dimmer or brighter than the fixed initial acquisition dynamic range were not included for analysis. We verified that shifting the acquisition window across fluorescence intensity ranges produced linear correlations throughout the range. In co-transfection of GFP and RFP experiments, cells that were non-fluorescent in either the green or red channel were not imaged, therefore the R² values for our co-transfection experiments are likely to be overestimates of the true R².

Image Analysis. Images were selected for analysis based on identification of single cells and low background. Images were adjusted for contrast and brightness only. Image analysis was performed blind to genotype. Fluorescence pixel intensities were measured in several regions of interest (ROIs) within the cell using a custom written program in MatLab (MathWorks, Natick, Mass.) or ImageJ. Average pixel intensities were calculated from three ROIs of 10×10 pixels for measurements within the cytoplasm and nucleus, or from five ROIs of 3×3 pixels for membrane and mitochondrial measurements. For Drosophila small lateral ventral neuron analysis, six ROIs of 6×6 pixels were measured from six neurons per lobe, and six animals per time point were chosen. All signal intensities were background subtracted from the average of three 10×10 ROIs surrounding the cell. We verified that RFP was still cyclically co-translated at later time points by analyzing red fluorescence intensities on Day 5 and 6 using a lower acquisition setting (FIG. 10c).

Electrophysiology. Standard whole cell voltage clamp was used to record potassium currents from HEK293 cells. During recordings, cells were maintained for 1-2 hours at 25° C. in extracellular solution consisting of 140 mM NaCl, 10 mM CaCl₂, 5 mM KCl, 10 mM HEPES, and 10 mM glucose at pH 7.4, 319 mOsm. Patch electrodes were pulled from standard wall borosilicate glass (BF150-86-10, Sutter instruments, Novato, Calif.) with 3-5 MΩ resistances. The intracellular pipette solution was 150 mM KCl, 2 mM MgCl₂, 1 mM CaCl₂, 2 mM EGTA, 20 mM HEPES, and 20 mM sucrose at pH 7.23, 326 mOsm. Whole cell potassium currents were low pass filtered at 5 kHz and measured using an Axopatch 200B amplifier (Axon instruments, Sunnyvale, Calif.), and recorded using a DigiData 1200 with pClamp9 software (Molecular Devices). All pipette and cell capacitances were fully compensated. Cells were held at −80 mV and then given +10 mV steps of 35 ms. The steady-state current elicited at +30 mV was used for analysis. Consistent cell capacitance, and membrane and access resistances were verified before and after recordings.

Statistical Analysis. Linear correlations were calculated by fitting the data to a simple linear regression model, with the coefficient of determination, R². We tested the null hypothesis that the variables were independent of each other and that the true R² value was 0. To test the confidence of our R² values for each experiment, we calculated the F statistic and its P value of the F-test on the regression model. We also used the permutation test to obtain a P value on the likelihood of obtaining our R² value by randomly shuffling the data and calculating a new R² value, repeated for one million runs (FIG. 7). Both approaches gave similar P values for all experiments.

To compare the R² values generated from PQRs to other conditions, we used the data from the fusion protein experiments as positive controls. We used the bootstrap method to generate a 95% confidence interval for the true R² value of the positive controls. We randomly chose 80% of the positive control data points to calculate a new R² value and repeated this for ten million runs, and used these simulated R² values to obtain upper and lower estimates of the positive control R² values (FIG. 7). All statistical analyses were performed using custom-written programs in MatLab (Mathworks).

Drosophila melanogaster Circadian Experiments. To generate the UAS-RFP-PQR-PER::YFP construct, PER::YFP was amplified from hs-PER::YFP, ligated with the RFP-PQR fragment, and inserted into the pUAST vector. Transgenic fly lines were created using P-element transgenesis (Bestgene Inc, Chino Hills, Calif.). The UAS-RFP-PQR-PER::YFP flies were crossed to the per-Gal4 driver line, P{GAL4-per.BS}3. Crosses were maintained at 25° C. in a 12 hour light-dark cycle incubator and newly eclosed F1 progeny were entrained for three days before collection. Six female flies were selected for each time point (6 AM and 6 PM, or zeitgeber time ZT0 and ZT12, respectively). Flies were fixed in 3.7% paraformaldehyde in 0.2M carbonate-bicarbonate buffer, pH=9.5 at 4° C. for 12 hours. Fly brains were then dissected, mounted on slides, and imaged using confocal microscopy.

Drosophila Dendritic Complexity Experiments. The pJFRC-20XUAS-IVS-RFPnls-PQR-cut construct was created by genomic extraction of the cut coding region from the fly UAS-cut (Grueber et al., 2003). The cDNA was ligated to RFPnls-PQR, and the resulting construct was cloned into the pJFRC7 vector. The transgenic fly w-; P{20XUAS-IVS-RFPnls-PQR-cut}attP was created by PhiC31 integrase-mediated transgenesis (Bestgene Inc). Homozygous flies w-; P{20XUAS-IVS-RFPnls-PQR-cut}attP, were crossed to homozygous w-; 221-Gal4, UAS-mCD8::GFP to selectively express RFPnls-PQR-cut in class I da neurons. Crosses were maintained at 18° C. and wandering third instar larvae were used for imaging. Larvae were dissected in phosphate-buffered saline and the anterior end, gut, tracheal tubes, and fat bodies were removed prior to imaging. Class I ddaE living neurons were imaged using a Fluoview FV1000 confocal laser scanning microscope (Olympus). Neuronal morphology was visualized using the membrane-bound mCD8::GFP and Cut protein levels were determined by ROI analysis of nuclear red fluorescence intensity. Complete dendritic arbors were reconstructed and the number of terminal branches and total dendritic length were computed using the NeuronJ plugin in Fiji.

Genome Editing using CRISPR-Cas9. Guide RNAs were designed as 20 bp DNA oligonucleotides and cloned into pX330 (Addgene 42230), and co-transfected with a circular PQR repair template using Lipofectamine 3000 (Life Technologies). All CRISPR-Cas9 guide RNAs were tested for activity using SURVEYOR Nuclease and SURVEYOR Enhancer S (Transgenomics) on extracted genomic DNA. Re-annealed products were analyzed on 4%-20% Novex TBE polyacrylamide gels (Life Technologies). Repair templates were constructed by placing PQR-XFP between homology arms specific to human, mouse, or fly RPL13A. The homology arms lacked the RPL13A promoter, which prevented expression of the PQR-XFP until in-frame genomic integration within an active coding gene. Left and right homology arms were 1.0 kb for the human genome, 1.5 kb for the mouse genome, and 700 bp for the Drosophila genome. Cellular fluorescence from PQRs was observed four days post-transfection.

Validation of PQR Genomic Insertion. Genotyping experiments were performed in experimental duplicate. Integration of PQR into the endogenous RPL13A or IgK genomic locus was validated by genomic DNA extraction six days post-transfection and genotyping using primers outside and within the homology arms of the repair template. The 5′ and 3′ ends were probed with two sets of primers and the endogenous RPL13a or IgK locus was PCR amplified. Restriction digests were then performed on PCR products at sites specific for PQR. All genomes were sequenced to identify the PQR and genomic junctions.

To verify that insertion of our PQR constructs into the endogenous RPL13A locus did not produce fusion protein products, we performed Western blots on manually enriched populations of the knock-in cell lines (FIG. 12). No fusion products were detected, and the enriched populations of knock-in cell lines were indistinguishable from wild-type cells with respect to phenotype and growth rate, and have been passaged multiple times. Finally, we also used quantitative PCR to verify that that the genome-edited cells produced RNA transcripts at similar levels to wildtype (FIG. 12).

Quantitative Real-Time PCR. For relative quantification of RPL13A and IgK mRNA levels from manually enriched stable cell lines, total RNA was extracted and purified using the PureLink RNA mini kit (Life Technologies) and genomic DNA was eliminated using DNaseI (New England Biolabs). Total RNA was reverse-transcribed with gene-specific primer cocktails (2 μM final concentration of each primer) using Superscript III reverse-polymerase (Life Technologies). This cDNA template was used for real-time PCR using the TaqMan Fast Advanced Mastermix (Life Technologies). Real-time PCR amplification was detected using the StepOnePlus Real-Time PCR System (Applied Biosystems) and cycle quantification values were calculated using the StepOne software. Experiments were performed in two to three experimental replicates with two technical replicates. Relative gene expression was determined using a ΔΔCq method. For relative quantification experiments, cycle quantification values were normalized to GAPDH in HEK293, N2A and 22c10 cells.

For absolute quantification of RPL13A and IgK mRNA levels from single cells, individual cells were imaged in drops of culture media on Teflon-coated glass slides before extraction and purification of total RNA using the TRIzol reagent (Life Technologies). Absolute quantification of RPL13A and IgK copy numbers was determined using standard curves generated with synthesized oligo standards containing the RPL13A and IgK target (sequences shown in Tables 4). Primers and double-quenched 5′-FAM/ZEN/IowaBlackFQ-3′ probes were purchased from Integrated DNA Technologies (Coralville, Iowa). All DNA and primer sequences used are shown in Table 4.

TABLE 4A
Nucleotide sequence and amino acid sequence of the CHYSEL peptide used in the Examples
		CHYSEL Oligo
CHYSEL peptides	CHYSEL DNA sequences	Peptide Sequences
vT2A_18aa	GAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAA	EGRGSLLTCGDVEENPGP
	TCCTGGACCT (SEQ ID NO: 7)	(SEQ ID NO: 2)
T2A_variant 1_Optimized	GAGGGACGCGGATCCCTGCTGACCTGCGGCGATGTGGAGGAGA	EGRGSLLTCGDVEENPGP
	ACCCCGGACCG (SEQ ID NO: 85)	(SEQ ID NO: 2)
T2A_variant 2_deoptimed	GAAGGTCGTGGTAGTCTACTAACGTGTGGTGATGTAGAAGAAAAT	EGRGSLLTCGDVEENPGP
	CCTGGACCT (SEQ ID NO: 86)	(SEQ ID NO: 2)
T2A_variant 3_deoptimed	GAAGGTCGTGGTAGTCTACTAACGTGTGGTGACGTCGAGGAAAA	EGRGSLLTCGDVEENPGP
	TCCTGGACCT (SEQ ID NO: 87)	(SEQ ID NO: 2)
T2A_mutant	GAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAA	EGRGSLLTCGDVEENAAP
	TGCGGCGCCT (SEQ ID NO: 88)	(SEQ ID NO: 95)
vP2A_30aa	GCTATGACTGTGATGGCATTTCAGGGGCCAGGT-	AMTVMAFQGPGATNFSLLKQAG
	GCCACTAACTTCTCCCTTTTAAAACAAGCAGGGGATGTTGAAGAA	DVEENPGP (SEQ ID NO: 96)
	AATCCCGGGCCC (SEQ ID NO: 89)
vP2A_19aa	GCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGA	ATNFSLLKQAGDVEENPGP
	GAACCCTGGACCT (SEQ ID NO: 4)	(SEQ ID NO: 1)
P2A_variant 1	GCGACGAATTTTAGTCTACTAAAACAAGCGGGTGATGTAGAAGAA	ATNFSLLKQAGDVEENPGP
	AATCCGGGTCCG (SEQ ID NO: 90)	(SEQ ID NO: 1)
P2A_variant 2	GCGACGAATTTTAGTCTACTAAAACAAGCGGGTGATGTAGAAGAA	ATNFSLLKQAGDVEENPGP
	AACCCTGGACCT (SEQ ID NO: 91)	(SEQ ID NO: 1)
P2A_variant 3	GCGACGAATTTTAGTCTACTGAAACAAGCGGGAGACGTGGAGGA	ATNFSLLKQAGDVEENPGP
	AAACCCTGGACCT (SEQ ID NO: 92)	(SEQ ID NO: 1)
P2A_variant 4	GCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGA	ATNFSLLKQAGDVEENPGP
	GAATCCGGGTCCG (SEQ ID NO: 93)	(SEQ ID NO: 1)
P2A_mutant	GCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGA	ATNFSLLKQAGDVEENAAP
	GAACGCGGCGCCT (SEQ ID NO: 94)	(SEQ ID NO: 1)

TABLE 4B
Nucleotide sequence of primers and probes used in the Examples
			Reverse-
			Transcription
Gene	Forward Primer	Reverse Primer	Primer	Probe
Human RPL13A	CTGGAGGTCAGTGATG	TGGAATGGTGTGGCAA
outside of homology	AGCA (SEQ ID NO: 97)	GTTA (SEQ ID NO: 98)
arms
Mouse Rpl13a	CGGGTTGCTAACCTGG	CAGTCTCCATCAAGGG
outside of homology	AATA (SEQ ID NO: 99)	GAAA (SEQ ID NO: 100)
arms
Drosophila RpL13A	TGAACCTCTCGGGACA	TGTGGATATGGTTGCA
outside of homology	CTTC (SEQ ID NO: 101)	TTCTG (SEQ ID NO:
arms		102)
Mouse IgK outside of	GGGGGAAAGGCTGCT	TAACTGGGGGAAGGG
homology arms	CATAA ((SEQ ID NO:	ACACT ((SEQ ID NO:
	103)	104)
RFP	ATGGTGTCTAAGGGCG	TTACTTGTACAGCTCG
	AAG (SEQ ID NO: 105)	TCCATG (SEQ ID NO:
		106)
GFP	TTGATGGAGCCAAAGA	GTACGTGTTCCGTAAG
	TGTG (SEQ ID NO: 107)	ACGG (SEQ ID NO: 108)
Human RPL13A	TGTTTGACGGCATCCC	CTGTCACTGCCTGGTA	CTGCTGGCCACATTTT	CTTCAGACGCACGACCTTGA
	AC (SEQ ID NO: 109)	CTTC (SEQ ID NO: 110)	ATGTC (SEQ ID NO:	GGG (SEQ ID NO: 112)
			111)
Mouse Rpl13a	TCCCTCCACCCTATGA	GTCACTGCCTGGTACT	GCAGCCCTGCTACTCA	ACGCCCCAGGTAAGCAAACTT
	CAAG (SEQ ID NO: 113)	TCC (SEQ ID NO: 114)	TTTTC (SEQ ID NO:	TCT (SEQ ID NO: 116)
			115)
IgK	AGTGGAAGATTGATGG	CTGTCTTTGCTGTCCT	GGTGGATTTCAGGGC	ACAAAATGGCGTCCTGAACAG
	CAGTG (SEQ ID NO:	GATCA (SEQ ID NO:	AACTA (SEQ ID NO:	TTGG (SEQ ID NO: 120)
	117)	118)	119)
Human GAPDH	GATCATCAGCAATGCC	GTCATGAGTCCTTCCA	GTACATGACAAGGTGC	TGGCCAAGGTCATCCATGACA
	TCCT (SEQ ID NO: 121)	CGATAC (SEQ ID NO:	GGCT	ACT (SEQ ID NO: 124)
		122)	(SEQ ID NO: 123)
Human RPL13A	CAAATACACAGAGGTC	CTTCGCCCTTAGACAC	CTGCTGGCCACATTTT	TGGTCGGAAGCGGAGCTACT
(Single Cell	CTCAAGA (SEQ ID NO:	CATAG (SEQ ID NO:	ATGTC (SEQ ID NO:	AACT (SEQ ID NO: 128)
experiments)	125)	126)	127)
Mouse Rpl13a	TGCAAGTTCACAGAGG	CTTCGCCCTTAGACAC	GCAGCCCTGCTACTCA	AGACTAAAATTCGTCGCTCCG
(Single Cell	TCC (SEQ ID NO: 129)	CATAG (SEQ ID NO:	TTTTC (SEQ ID NO:	CTTCC
experiments)		130)	131)	(SEQ ID NO: 132)
IgK Constant Region	TCACAAGACATCAACTT	TCCACGTCTCCAGCCT	GGTGGATTTCAGGGC	AGCTCCGCTTCCACACTCATT
	CACCC (SEQ ID NO:	GCT (SEQ ID NO: 134)	AACTA (SEQ ID NO:	CC (SEQ ID NO: 136)
	133)		135)
Drosophila RpL13A	CGACGTCAGCTAGGAG	TGAAATTGGTTTGTGC
knock-in animal	TGTG (SEQ ID NO: 137)	CTACC (SEQ ID NO:
		138)
RFP knock-in animal	GGCCACCTGATCTGCA	TTCTGCTGCCGTACAT
	AC (SEQ ID NO: 139)	GAAG (SEQ ID NO: 140)

TABLE 4C
Nucleotide sequence of the oligonucleotides used in the Examples
Gene   Synthesized Oligo Sequence
Mouse  AGGCAGAAAAGAATGTGGAGAAGAAAATCTGCAAGTTCACAGAGGTCCTCAAGACCAACGGACTCCTGGTGGGAAGCGGA
Rpl13a GCGACGAATTTTAGTCTACTAAAACAAGCGGGTGATGTAGAAGAAAACCCTGGACCT (SEQ ID NO: 141)
IgK    GACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGAATGAGTGTGGA
       AGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTGCTAGCATGGTGAGCAAG
       GGCGAGGAGGATAACATGGCCTCTCTC (SEQ ID NO: 142)
Human  TACGGAAACAGGCCGAGAAGAACGTGGAGAAGAAAATTGACAAATACACAGAGGTCCTCAAGACCCACGGACTCCTGGTCGGA
RPL13A AGCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCTATGGTGTCTAAGGGCGAAGA
       GCTGATTAAGGAGAACATGCACATGAAGCTGTACATGGAGGG (SEQ ID NO: 143)

Example II

Ratioing Using the Protein Quantification Reporter Linker (PQR linker)

The experimental procedures associated with this example were presented in Example I.

We modified and tested different CHYSEL sequences for efficient and stoichiometric separation of the upstream and downstream genes and identified different sequences for use in Drosophila cells and vertebrate cells. We first screened for CHYSEL sequences that produce reliable separation of the upstream and downstream protein (FIG. 2). We created different CHYSEL sequences for use in Drosophila and vertebrate cells, taken from different RNA viruses and modified, and then codon de-optimized at specific residues (FIG. 2 and Example I). Thus, we collectively called these different DNA constructs for Drosophila and for vertebrate use, Protein Quantitation Reporters (PQRs).

Next, we tested the stoichiometric ratio and linear relationship between different genes separated by our PQRs at the single cell level. First, we quantified fluorescence intensities in Human Embryonic Kidney 293 (HEK293) cells expressing a fusion protein of one molecule of green fluorescent protein (GFP) attached to one molecule of Red Fluorescent Protein (RFP) by a mutated PQR linker (FIG. 3). Because fluorescence intensity is directly proportional to the concentration of fluorescent molecules over several orders of magnitude, particularly at physiological concentrations (mg per mL) (Furtado and Henry, 2002) (FIG. 1c), we measured the fluorescence output (i.e., brightness) of a cell to quantitate the ratio of GFP to RFP molecules. We found that green and red fluorescence intensities in cells expressing GFP::RFP were linearly correlated with a coefficient of determination, R²=0.74 (n=74 cells, P<0.001) (FIG. 3a). Co-transfection of GFP and RFP into cells produced green and red fluorescence intensities that had a weak covariance, with R²=0.37 (n=59 cells, P<0.001) due to differences in uptake, gene expression, and protein expression of GFP- versus RFP-encoding plasmids (FIG. 3b). Co-transfection of plasmids is a common technique used for qualitative determination of protein co-expression, where the amount of DNA for each plasmid is titrated to a desired expression level, and it is then incorrectly assumed that the brightness of the cell corresponds to the expression level of the co-transfected plasmid(s) (FIG. 3b). When we expressed GFP and RFP separated by a PQR sequence in cells, we found that the green and red fluorescence intensities were correlated with an R²=0.78 and 0.66 for GFP-PQR-RFP (n=77 cells, P<0.001) and RFP-PQR-GFP (n=77 cells, P<0.001), respectively (FIGS. 3c, d). These R² values for PQR constructs were within the 95% confidence interval for the R² value for the GFP::RFP fusion protein, whereas the co-transfection of GFP and RFP R² value was outside the 95% confidence interval (Example I and FIG. 7). These results demonstrate that a PQR can produce stoichiometric ratios of proteins indistinguishable from fusing a fluorescent reporter. These PQR results were also not due to the incomplete separation of the upstream and downstream genes, creating a subpopulation of GFP and RFP fusion product (FIG. 2). To further determine whether genes separated by a PQR produced spatially separated proteins, we expressed spectrally-distinct fluorophores with different subcellular localization signals, each separated by a PQR sequence in a single, polycistronic strand (YFPmito-PQR-CFPnls-PQR-RFP, FIGS. 8a-c). Fluorescence intensities of different colors in mitochondrial, nuclear, or cytoplasmic compartments were linearly correlated (ranging from R²=0.54 to 0.69 for different organelles, n=40 cells, P<0.001), and intensities for the non-expected fluorophores were not detectable above background, confirming that fusion proteins were not formed and localized to inappropriate cellular compartments. This also demonstrates that stoichiometric production of proteins is maintained for polycistronic mRNAs using PQRs, allowing for protein quantification in multiple regions of interest using different subcellular localization signals.

PQR can relate cellular phenotype as a function of protein concentration. To determine whether PQR fluorescence intensity could correlate with a cellular phenotype directly related to protein concentration, we measured ion channel concentrations using whole cell patch clamp electrophysiology. We expressed the Drosophila Shaker potassium channel with a GFP molecule embedded within the inactivation domain (Batulan et al., 2010) separated by PQR-RFP (ShakerGFP-PQR-RFP) in HEK293 cells (FIG. 3e). Measurements of K⁺ channel current density compared to green fluorescence intensity of the cell membrane produced a linear correlation of R²=0.73 (n=28 cells, P<0.001) (FIG. 3f). Current density as a function of red fluorescence intensity had a correlation of R²=0.55 (P<0.001), and green to red fluorescence correlation was R²=0.84 (P<0.001) (FIGS. 3g-h), indicating that the steady-state ratio between RFP and a membrane protein four times larger and with several fold slower turnover maintained a linear relationship across expression ranges (coefficient of variation for current was 0.34) (Corish and Tyler-Smith, 1999; Zhao et al., 1995). We performed these electrophysiological and image analyses using different fluorescence microscopy methods and found strong linear correlations amongst all excitation methods (e.g., metal-halide lamp, mercury vapor lamp, lasers), fluorophores, and microscopy methods tested (ranging from R²=0.90 to 0.94 between different methods) (FIG. 8d). This is expected because the linear relationship between concentration of a fluorophore and its brightness (FIG. 1c) will be maintained regardless of excitation source, fluorophore, or emission detection method. This demonstrates that all standard fluorescence microscopy methods can be used with this technique.

To demonstrate the applicability of this technique in single neurons in animals, we used the predictable and quantitative changes in protein amounts that occur in the circadian system (FIG. 4a). The transcription factor Period, or PER, controls the circadian rhythms of the Drosophila brain, and PER protein levels cycle every 24 hours as it is synthesized, shuttled into and out of the nucleus, and degraded in the proteasome. To measure cyclic changes in fluorescence intensity in single cells, we used a fusion protein of PER and Yellow Fluorescent Protein (PER::YFP) separated by PQR with RFP. We used period-Gal4 to drive expression of the UAS-PER::YFP-PQR-RFP construct within small lateral ventral neurons of the Drosophila brain (FIG. 4a). We found that yellow fluorescence intensities cycled with a 24 hour periodicity without increasing beyond a fixed point, as the PER::YFP fusion protein was continually formed and destroyed (FIG. 4b). RFP has a slightly longer half-life (26 hours) than PER, thus as RFP was co-translated and separated from the PER::YFP, we observed parallel production and degradation at early time points. However, the red fluorescence intensities eventually increased cyclically over several days until it saturated the fixed acquisition settings, set at the initially low red fluorescence intensities (FIG. 4b).

We next used PQR to determine a quantitative relationship between protein amount and cellular phenotype in single living cells. Drosophila dendritic arborization (da) neurons can be classified into four groups (class I, II, III, and IV) based on their dendritic arbor complexity, and the transcription factor Cut has been implicated in regulating this complexity in a dosage-dependent manner. However, it is not clear how Cut protein levels regulate neurite outgrowth. For example, the transcription factor may act as a binary switch, or have a linear relationship with dendritic growth. Because da neurons are relatively large cells (FIG. 4c) and we surmised that as a transcription factor, low levels of Cut would produce significant phenotypes, we used a PQR with a nuclear localization signal (RFPnls) to sequester the fluorophore and enhance the signal. We selectively expressed UAS-RFPnls-PQR-cut in class I da neurons using the 221-Gal4, UAS-mCD8::GFP line. We measured red fluorescence within the nucleus and used GFP to image the dendritic morphology to quantify the total dendritic arbor length and number of terminal branches (FIG. 4c). We found that dendritic arbor complexity (number of dendritic terminals or total dendritic length) increases logarithmically with Cut protein levels until the dendritic branching effect was saturated (FIG. 4d). These results indicate that Cut regulates dendritic arbor complexity in a concentration-dependent non-linear manner.

We next sought to insert PQRs into endogenous genomic loci to create a polycistronic mRNA that would preserve regulatory elements, such as the mRNA untranslated regions (UTR) (FIG. 5a). We used Clustered Regularly Interspaced Short Palindromic Repeats-Cas9 (CRISPR-Cas9) genome editing to generate custom RNAs that guide Cas9 nuclease to create a double-strand break at a specific genomic locus. DNA double-strand breaks within a cell can be repaired through homologous recombination, and in the presence of an exogenous repair template containing DNA sequences of interest flanked by homologous sequence arms, foreign sequences can be recombined into the genome. We generated repair templates to insert a PQR after the protein coding sequence of a gene, but before the final stop codon and 3′ UTR, to produce a single RNA strand encoding the endogenous protein of interest and a fluorescent reporter (FIG. 5a). We inserted PQRs with RFP or blue fluorescent protein with a nuclear localization signal (BFPnls) within the endogenous RPL13A genomic locus in human, Drosophila, and mouse genomes using HEK293, Kc, and Neuroblastoma-2A cells, respectively (FIGS. 5b-d).

Using these genome-edited cells, we then wanted to examine the relationship between absolute mRNA transcript numbers and protein amount in the same cell. We combined PQR of endogenous protein production with single cell quantitative PCR (FIG. 6). We first imaged a live cell expressing a PQR and then lysed the cell to extract and measure its PQR mRNA transcripts (FIG. 6a). Using our HEK293 cell line carrying a PQR-RFP reporter at the endogenous RPL13A locus (FIG. 5b), we found that the number of mRNA molecules ranged from 50 to 570, and RPL13A relative protein amounts as measured by RFP fluorescence intensity, clustered between 200 to 800 arbitrary units, resulting in no correlation between RPL13A mRNA versus protein amounts (R²=0.03; n=22) (FIG. 6b-d). As a comparison, we inserted a PQR-GFP reporter into the immunoglobulin kappa (κ) light chain genomic locus, IgK, in the mouse monoclonal antibody cell line, 22c10 (FIG. 6e). As expected, these 22c10 cells produced a large amount of IgK mRNA ranging from 1 500 to 180 500 molecules in a single cell, despite being derived from a single clonal cell (FIGS. 6f, g). The green fluorescence intensity distribution also varied widely, which produced a weak correlation between IgK transcript number and IGK protein amount (R²=0.22, n=36) (FIG. 6h). To confirm that our fluorescence intensity distributions and correlations were not due to differences between the fluorophores, cell types, or procedure, we first swapped the PQR fluorophores from the Rpl13a and IgK genes to create Rpl13a-PQR-GFPnls and IgK-PQR-RFP. Next, we used CRISPR-Cas9 genome-editing on both of these genes within a single cell line to create double knock-in cells (FIG. 9). By measuring green fluorescence in the nucleus and red fluorescence in the cytoplasm within a single 22c10 cell, and then quantifying its Rpl13a and IgK mRNA amounts, we verified that the mRNA expression of these genes is a poor predictor of actual protein translation. These results using both mouse and human genes confirm previous studies demonstrating the poor correspondence between mRNA expression and actual protein production.

The technique we describe here, Protein Quantitation Ratioing, uses standard fluorescence imaging available through multiple microscopy methods. PQR is fast, has sensitivity at single cell resolution, and can be performed with time-lapse in living cells. Using a cell's brightness as a readout for the protein expression level of a gene, PQR can have a wide range of applications in cell biology to quantitatively measure relationships between phenotypes and protein levels.

The PQR technique quantifies steady-state protein levels within a cell, and differences in kinetics of the upstream and downstream proteins (e.g., folding, maturation, or turnover rates) will change the slope of the linear relationship, but the fluorescence will still be proportional to the number of molecules translated (FIG. 1b). For example, the Drosophila Shaker K⁺ channel is homo-tetrameric (i.e., four molecules are required for a single functional channel), has a turnover rate of several days, but has complex and comparatively rapid internalization and insertion rates on timescales of minutes to hours. Our PQR results using the Shaker K⁺ channel demonstrate that the technique can be used even for complicated membrane proteins with slow degradation rates. In addition, protein concentration is predominantly controlled by translation, with very small contribution (<5%) from protein stability and degradation. To model how differences in protein dynamics might affect PQR measurements, we simulated two cells expressing PQRs that exhibited different kinetics of a protein of interest (FIG. 10a). We found that differences in protein turnover did not adversely affect PQR accuracy, with >85% of cases producing at least 90% accurate quantification, across tens of thousands of proteins with randomly varying kinetics (FIG. 10). This is because PQR measurements are ratiometric between two cells rather than absolute measurements of protein abundance, and because the CHYSEL mechanism forces not only the identical ON rate of the protein of interest, but also the exact protein amount being produced. This creates a regularly resetting mechanism for the PQR fluorophore to match the protein of interest kinetics. Experimentally, we used the circadian system as an extreme example of very tightly regulated gene expression, with precisely controlled mRNA production and mRNA degradation, and protein production and degradation. Our PQR measurements could integrate the cyclic production of PER protein until the PQR fluorescence intensities saturated (FIG. 4b). However, cyclic changes in PER protein can still be accurately measured at any arbitrary later time point by resetting the acquisition setting for PQR fluorescence (FIG. 10c), demonstrating the robust sensitivity of the PQR technique. More precise spatial and temporal measurements of protein kinetics may be obtained through the use of photoswitchable molecules to allow for subcellular activation and quantitative imaging of newly synthesized fluorescent molecules. Although the PQR technique quantifies protein amounts indirectly, it is a similarly indirect measurement as quantitative immuno-blots and quantitative PCR. Currently, the only alternatives to PQR are quantitative immuno-blots and protein assays, which require isolation of large amounts of heterogeneous starting material.

For our positive controls, we fused a fluorescent protein to a protein of interest to track and quantitate protein amounts. However, unlike a physical tag, PQR uses a genetic tag separated during protein synthesis, leaving only ˜20 amino acids on the carboxy terminus of the upstream protein and a single proline at the start of the downstream protein. A fusion protein must be expressed at high enough levels to detect, and be accessible for analysis (e.g., it may be membrane-associated or may be secreted), and any modification can interfere with protein stability, activity, or function (e.g., N-terminus and C-terminus additions can affect Type I and Type II transmembrane proteins, or alter intracellular signaling). Using the integral membrane protein Shaker K⁺ channel, we verified that placement of the Shaker gene upstream or downstream of the PQR sequences did not affect its membrane insertion or properties (FIG. 8e). Separation of the PQR to different locations than the protein of interest allows for easier quantification of genes expressed at low levels (FIG. 4c), where the PQR can be sequestered within the nucleus or nucleolus or for large or complex cells such as neurons (FIG. 4c), or for quantification of transmembrane and secreted proteins, such as the production of antibodies. For example, the genomic organization of vertebrate antibodies joins upstream variable exons to a final 3′ constant exon and insertion of a PQR between the coding sequence and the 3′ UTR will allow for quantification of antibody production in all cells that synthesize the specific antibody type.

The RPL13A gene encodes for Ribosomal Protein L13A and is expressed in every cell in all eukaryotes at moderately high levels (FIG. 6b), and is commonly used as a housekeeping gene for normalization in quantitative DNA and protein measurements. Therefore, quantitation of endogenous RPL13A protein levels in single cells can be used as a measure of an individual cell's overall transcriptional and translational status (FIGS. 5, 6). Quantifying RPL13A fluorescence levels in a second channel (e.g., RFP or BFPnls) allows for normalization across cells or experiments, and for optical effects such as spherical aberration, optical distortions, and imaging depths during in vivo imaging. Thus, using this approach the relative levels of any protein of interest can be determined across conditions using the ratio of fluorescence between the protein of interest normalized to RPL13A fluorescence.

Quantification of endogenous proteins using PQR does not necessarily require the generation of knock-in organisms. For example, efficient genome editing of post-mitotic neurons transfected with the CRISPR-Cas9 system has been demonstrated using biolistic transfection and in utero electroporation. This will allow for PQR of endogenous proteins within specific cells in vivo, for example by transfection of CRISPR-Cas9 for homologous recombination of PQR constructs within neurons. The Protein Quantitation Ratioing technique has broad expansion possibilities, such as measuring protein production in single cells over time for drug screening, quantitation of endogenous protein levels in single cells in vivo, normalization across experiments and optical effects using the ratio of RPL13A levels, and allowing a wide range of quantitative experiments examining gene to phenotype relationships.

Example III

PQR Optimization

The experimental procedures used in this example are presented in Example I.

In order to determine how to optimize PQR, we first assessed whether the tri-peptide GSG was necessary for performing protein quantification, we compared the cleavage of a PQR linker bearing and lacking the GSG tri-peptide (refer to Table 5 for a description of the sequences used).

TABLE 5
Description of wild-type and GSG-modified P2A and T2A peptides as well
as wild-type and codon-optimized DNA sequences encoding for such peptides.
SEQ ID
NO	Sequence	Description
1	ATNFSLLKQAGDVEENPGP	Native P2A peptide from Teschovirus-1
3	GSGATNFSLLKQAGDVEENPGP	P2A peptide from teschovirus-1 to which a
		GSG peptide has been added at the N-
		terminus
4	GCT ACT AAC TTC AGC CTG	DNA sequence encoding the native P2A
	CTG AAG CAG GCT GGA GAC	peptide (SEQ ID NO: 1) and corresponding
	GTG GAG GAG AAC CCT GGA	to the viral RNA sequence (i.e., not-codon
	CCT	optimized)
5	GGA AGC GGA GCC ACA AAC	DNA sequence encoding the modified P2A
	TTC AGT CTC CTG AAA CAG	peptide (SEQ ID NO: 3). The sequence
	GCA GGC GAT GTG GAG GAG	corresponds to a 100% (excluding the GSG
	AAT CCC GGC CCA	tri-peptide encoding sequence, identified in
		bold) or 86% (including the GSG tri-peptide
		encoding sequence) codon-optimized for the
		mouse model.
2	EGRGSLLTCGDVEENPGP	Native T2A peptide from Thosea asigna
		virus
6	GSGEGRGSLLTCGDVEENPGP	Modified T2A peptide to which a GSG
		peptide has been added at the N-terminus
7	GAG GGC AGA GGA AGT CTG	DNA sequence encoding the native T2A
	CTA ACA TGC GGT GAC GTC	peptide (SEQ ID NO: 2) and corresponding
	GAG GAG AAT CCT GGA CCT	to the viral RNA sequence (i.e., not-codon
		optimized)
8	GGA AGC GGA GAG GGG AGA	DNA sequence encoding the modified T2A
	GGG TCT CTG CTG ACC TGC	peptide (SEQ ID NO: 6) and corresponding
	GGG GAT GTC GAG GAG AAC	to a 100% (excluding the GSG tri-peptide
	CCC GGC CCC	encoding sequence, identified in bold) or
		86% (including the GSG tri-peptide encoding
		sequence) codon-optimized for the
		Drosophila melanogaster model.

As shown on FIG. 14, the presence of the GSG tri-peptide is required for proper separation of the two poly-proteins and for proper protein function (FIG. 14A). Without the proper separation, the resulting fusion protein product that is produced is often non-functional and degraded by the cell (FIG. 14B).

Next, we wanted to determine if codon optimization of the corresponding DNA sequences encoding the PQR peptides could further increase cleavage of the two proteins. To do so, native viral DNA sequences and codon optimized DNA sequences (but both with GSG-modification, refer to table 5 for a complete description) were first compared for their ability to produce separate proteins and limit the presence of a fusion protein product. We found that both the original viral sequence and the codon optimized forms resulted in a fraction of un-separated, fusion product (FIG. 13). To our surprise however, we also found that codon optimization sometimes created a larger proportion of un-separated product, meaning the codon optimization could be worse for using a PQR for protein quantitation in single cells.

Without wishing to be bound by theory, it was hypothesized that codon optimization sometimes sped up the ribosomal activity during translation causing it to ignore the separation event between the glycine and proline bond of the CHYSEL peptide (Zhou et al. 2011; Novoa and Ribas de Pouplana 2012). Slowing down the ribosome using unfavored codons should therefore increase separation of the PQR sequence. Consequently, we tested the variations of the P2A and T2A sequences presented in Table 6 to determine their usefulness in protein ratioing.

TABLE 6
DNA sequences encoding the P2A (SEQ ID NO: 1) or T2A (SEQ ID NO: 3)
peptides. Codons in bold have been de-optimized to for the mouse
model (i.e., the codon least favored in the host has been selected).
Codons underlined with a double line have been optimized for the
Drosophila melanogaster model (i.e., the codon most favored in the
host has been selected). Underlined codons have been mutated
to code for alanine.
SEQ
ID NO:	Sequence	Description
4	GCT ACT AAC TTC AGC CTG	Wild-type viral DNA sequence encoding
	CTG AAG CAG GCT GGA GAC	SEQ ID NO: 1
	GTG GAG GAG AAC CCT GGA
	CCT
9	GGA AGC GGA GCG ACG AAT	Variation 1 of SEQ ID NO: 4. This sequence
	TTT AGT CTA CTA AAA CAA	corresponds to a 100% (excluding the GSG
	GCG GGT GAT GTA GAA GAA	tri-peptide encoding sequence) or 86%
	AAT CCG GGT CCG	(including the GSG tri-peptide encoding
		sequence) codon-deoptimized sequence.
10	GGA AGC GGA GCG ACG AAT	Variation 2 of SEQ ID NO: 4. This sequence
	TTT AGT CTA CTA AAA CAA	corresponds to a 80% (excluding the GSG
	GCG GGT GAT GTA GAA GAA	tri-peptide encoding sequence) or 68%
	AAC CCT GGA CCT	(including the GSG tri-peptide encoding
		sequence) codon-deoptimized sequence.
11	GGA AGC GGA GCG ACG AAT	Variation 3 of SEQ ID NO: 4. This sequence
	TTT AGT CTA CTG AAA CAA	corresponds to a 50% (excluding the GSG
	GCG GGA GAC GTG GAG GAA	tri-peptide encoding sequence) or 45%
	AAC CCT GGA CCT	(including the GSG tri-peptide encoding
		sequence) codon-deoptimized sequence.
12	GGA AGC GGA GCT ACT AAC	Variation 4 of SEQ ID NO: 4. This sequence
	TTC AGC CTG CTG AAG CAG	corresponds to a 21% (excluding the GSG
	GCT GGA GAC GTG GAG GAG	tri-peptide encoding sequence) or a 18%
	AAT CCG GGT CCG	(including the GSG-tripeptide encoding
		sequence) codon-deoptimized sequence.
13	GGA AGC GGA GCT ACT AAC	Mutant of SEQ ID NO: 4 in which the
	TTC AGC CTG CTG AAG CAG	underlined codons have been mutated to
	GCT GGA GAC GTG GAG GAG	code for an alanine. This sequence serves
	AAC GCG GCG CCT	as a control in which none of the fusion
		proteins are cleaved.
6	GAG GGC AGA GGA AGT CTG	Wild-type viral DNA sequence encoding
	CTA ACA TGC GGT GAC GTC	SEQ ID NO: 3
	GAG GAG AAT CCT GGA CCT
14	GGA AGC GGA GAA GGT CGT	Variation 1 of SEQ ID NO: 6. This sequence
	GGT AGT CTA CTA ACG TGT	corresponds to a 60% (excluding the GSG
	GGT GAT GTA GAA GAA AAT	tri-peptide encoding sequence) or a 52%
	CCT GGA CCT	(including the GSG-tripeptide encoding
		sequence) codon-deoptimized sequence.
15	GGA AGC GGA GAA GGT CGT	Variation 2 of SEQ ID NO: 6. This sequence
	GGT AGT CTA CTA ACG TGT	corresponds to a 45% (excluding the GSG
	GGT GAC GTC GAG GAA AAT	tri-peptide encoding sequence) or a 38%
	CCT GGA CCT	(including the GSG-tripeptide encoding
		sequence) codon-deoptimized sequence.
16	GGA AGC GGA GAG GGA CGC	Variation 3 of SEQ ID NO: 6.
	GGA TCC CTG CTG ACC TGC	This sequence corresponds to a 60%
	GGC GAT GTG  GAG GAG AAC	(excluding the GSG tri-peptide encoding
	CCC  GGA CCG	sequence) or a 52% (including the GSG-
		tripeptide encoding sequence) codon-
		optimized sequence. The codons underlined
		with a double line have been optimized by
		the software bestgene

The DNA sequences presented in Table 6 were introduced in HEK 293 cells using lipofectamine (life technologies Inc.) for the PQR sequences from P2A-based variants, and Drosophila S2 cells for the PQR sequences from T2A-based variants. We then tested each sequence for how much fusion product each variation would create, using immuno-blots (FIG. 7) to detect the fusion product. The actin and GFP bottom and middle row blots, respectively are controls to demonstrate that the separated product does form. The untransfected columns are used as negative controls. The P2A mutant and T2A mutant is used as a positive control to indicate where a fusion protein product will occur (what size the protein should run at on the immuno-blot). As shown on FIG. 7a, P2A variation 3 and T2A variation 2 produce the least amount of fusion product, close to background levels of the untransfected cell column. The quantitation of the results presented in FIG. 7a is shown in FIG. 7b.

As indicated in FIG. 15, stochoimetric cleavage have been achieved when using a sequence derived from the F2A peptide (SEQ ID NO: 17 to which as GSG peptide sequence has been added) as a PQR between the GFP and RFP reporter proteins. The PQR-encoding sequence was the following: GGT TCT GGT GCT CCT GTC AAA CAA ACT CTT AAC TTT GAT TTA CTC AAA CTG GCT GGG GAT GTA GAA AGC AAT CCA GGT CCA (SEQ ID NO: 82).

Example IV

In Vivo PQR

A knock-in animal with a protein quantitation reporter and fluorescent protein (PQR-XFP) integrated into the Ribosomal Protein L13A (RPL13A) locus can be an invaluable resource for biologists. It allows the quantification of RPL13A expression in any cell type and developmental stage of the animal. The PQR RPL13A fluorescence output represents the relative levels of RPL13A protein expression, and this can be used as a standard reference for normalization during in vivo imaging or to normalize a single cell's transcriptional and translational states. The knock-in PQR-XFP can be stably maintained in the RPL13A locus and passed on from generation to generation provided that the knock-in is genetically stabilized and heritable.

To create a knock-in Drosophila expressing PQR-RFPnols at the RpL13A genomic locus, we employed the strategy of first creating a transgenic fly expressing the guide RNA (gRNA) taregeting the RpL13A locus to be crossed to another fly expressing the Cas9 nuclease within the embryos (Port et al., 2014). The subsequent combination of the customized gRNA and Cas9 nuclease in the offspring, both expressed only in the embryo stage, forms the active CRISPR-Cas9 complex to perform genome editing at the the specific locus. The repair template containing the edited PQR-RFP locus is injected into these embryos, and are screened for positive results later in development and in their offspring to ensure germlie transmission.

To create the gRNA transgenic flies, a U6:3gRNA plasmid was first constructed and its genome targeting was verified in Drosophila Kc cells lines. High quality DNA plasmid was then prepared and sent to a transgenic service (The Bestgene, Inc) for embryo injections to create the transgenic flies. These gRNA flies were then crossed to nanos-cas9 flies, and embryos expressing the two components gRNA and Cas9 were collected for injection with the circular DNA repair template, RPL13A-PQR-RFPnols (Bestgene, Inc). After reaching adulthood, these G0 flies were crossed to one another and the resulting F1 larvae with red nucleoli in all cells were identified and isolated using a standard epi-fluorescence microscope (FIG. 16). The knock-in was verified by genotyping and Sanger sequencing (FIG. 16), as described above.

We have characterized the pattern of red fluorescent nuclei in living and dissected third instar larvae. Red nuclei were observed throughout the entire animal with varying degrees of fluorescent intensities between different tissues, implying different levels of cellular transcription and translation. For cells undergoing rapid proliferation during development, such as the body wall tissue and the gut, the fluorescence intensities are significantly stronger than those from post-mitotic cells, like neurons (FIG. 16). For cells of the same type, the variation in fluorescence intensity is considerably smaller than compared to cells coming from different tissues.

While the invention has been described in connection with specific embodiments thereof, it will be understood that the scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

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Claims

1. A protein quantitation reporter linker nucleic acid molecule for quantifying a protein of interest in a host cell, the protein quantitation reporter: (i) encoding a cleavable peptide having the amino acid sequence of SEQ ID NO: 1, 2 or 17; and(ii) being a nucleic acid molecule having the nucleic acid sequence in which at least 50% of the codons encoding the amino acid sequence of SEQ ID NO: 1, 2 or 17 are de-optimized in function of the host cell.

2. The protein quantitation reporter linker nucleic acid molecule of claim 1 being a deoxyribonucleic (DNA) molecule.

3. The protein quantitation reporter linker nucleic acid molecule of claim 1, wherein at least 80% of the codons are de-optimized in function of the host cell.

4. A vector for quantifying a protein of interest in a host cell, said vector comprising a first nucleic acid molecule encoding the protein quantitation reporter linker nucleic acid molecule of claim 1.

5. The vector of claim 4, further comprising a second nucleic acid molecule encoding a reporter protein operatively linked to the first nucleic acid molecule so that the first nucleic acid molecule and second nucleic acid molecule are transcribed as a single messenger RNA transcript from the vector.

6. The vector of claim 5, wherein the reporter protein is selected from the group consisting of a fluorescent protein, an antibiotic-resistance protein, an immunoglobulin protein, an ion channel, a transcription factor, a ribosomal protein, an enzyme and a receptor.

7. The vector of claim 6, wherein the fluorescent protein is selected from the group consisting of a green-fluorescent protein (GFP), a red fluorescent protein (RFP), a yellow fluorescent protein (YFP), a blue fluorescent protein (BFP) and a cyan fluorescent protein (CFP).

8. The vector of claim 4, further comprising a third nucleic acid molecule encoding a protein of interest operatively linked to the first nucleic acid molecule so that the first nucleic acid molecule and the third nucleic acid molecule are transcribed as a single messenger RNA transcript from the vector.

9. The vector of claim 4, further comprising a second nucleic acid molecule encoding a reporter protein and a third nucleic acid molecule encoding a protein of interest, wherein the second nucleic acid molecule and the third nucleic acid molecule are operatively linked to the first nucleic acid molecule and wherein the first nucleic acid molecule is located between the second nucleic acid molecule and the third nucleic acid molecule, so that the that the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule are transcribed as a single messenger RNA transcript from the vector.

10. The vector of claim 9, wherein the second nucleic acid molecule is upstream of the third nucleic acid molecule.

11. The vector of claim 9, wherein the second nucleic acid molecule is downstream of the third nucleic acid molecule.

12. A method for quantifying a protein of interest in a host or a host cell, said method comprising (i) expressing the vector of claim 9 in the host or the host cell so as to cause the generation of a nucleic acid transcript encoding a poly-protein, wherein the poly-protein comprises the protein of interest, a cleavable peptide and a reporter protein and wherein the cleavable peptide can be cleaved during the translation of the nucleic acid transcript to generate a cleaved reporter protein and (ii) measuring a signal associated with the cleaved reporter protein to quantify the protein of interest.

13. The method of claim 12, wherein the reporter protein is a fluorescent protein and step (ii) further comprises determining the fluorescence associated with the cleaved reporter protein.

14. The method of claim 12, wherein the host cell is a living cell.

15. The method of claim 12, wherein the host cell is a single cell.

16. The protein quantitation reporter linker nucleic acid molecule of claim 1, wherein the cleavable peptide further comprises a GSG tripeptide.

17. The protein quantitation reporter linker nucleic acid molecule of claim 1 encoding the amino acid sequence of SEQ ID NO: 2 and having the nucleic acid sequence of SEQ ID NO: 86 or 87.

18. The protein quantitation reporter linker nucleic acid molecule of claim 1 encoding the amino acid sequence of SEQ ID NO: 1 and having the nucleic acid sequence of SEQ ID NO: 90, 91 or 92.

19. The protein quantitation reporter linker nucleic acid molecule of claim 16 having the nucleic acid sequence of SEQ ID NO: 82.

20. The protein quantitation reporter linker nucleic acid molecule of claim 16 having the nucleic acid sequence of SEQ ID NO: 83.

21. The protein quantitation reporter linker nucleic acid molecule of claim 16 having the nucleic acid sequence of SEQ ID NO: 84.