Patent Grant
11414454
A sequence listing submitted in computer readable format is hereby incorporated by reference. The computer readable file is named P265260_WO-US_01-504865-00008_SL.txt, is 51,110 bytes in size, created on Nov. 5, 2019.
The disclosed processes, methods, and systems are directed to peptide sequences useful in expression, identification, and isolation of recombinant proteins and peptides.
Much of bio-medical research relies on the ability to identify, express, engineer, isolate, and analyze proteins in a clinical or research laboratory setting. In some cases, this requires a large array of different methods, kits, and reagents. While recombinant proteins are useful in analyzing a protein's function by making mutations in its sequence, it must be isolated and purified in order to test that function. There are a variety of reagents and systems for purifying proteins, but existing methods have important disadvantages. To minimize these disadvantages researchers are required to use multiple techniques, which result in increased costs and time.
There is a need for improved compositions, methods, systems, and kits for enhancing the expression, isolation, and identification of proteins, especially recombinant/engineered proteins.
The present disclosure is directed to compositions, proteins, nucleic acids, methods, and systems for purification and/or detection of recombinant proteins. In many embodiments, a Ribose Binding Protein is separated at or near its carboxyl end to generate two fragments that bind specifically, and with high affinity. When one or the other fragment is immobilized to a solid support, this specific interaction is robust and is able to withstand exposure to a wide range of pH environments. The disclosed interaction is also stable in a variety of denaturing conditions. The interaction may be further stabilized by addition of D-ribose. Also disclosed is a system that enhances recombinant protein expression and solubility.
The disclosed compositions, proteins, nucleic acids, methods, and systems are novel, non-obvious, and have great and varied utility. For example, the disclosed compositions may be useful in creating a variety of affinity purification resins, as well as various applications involving the expression, purification, or isolation of tagged recombinant proteins, including without limitation western blots, ELISAs, immunocytochemistry, etc.
Disclosed herein are compositions, methods, systems, and kits useful in the expression, identification, and isolation/purification of engineered proteins. In some embodiments, a two-part peptide tag system is disclosed that is useful for affinity purification and/or specifically identifying tagged proteins. The system is also useful in aiding solubility and expression of recombinant proteins while also providing a tag for identifying and isolating/purifying the recombinant protein. The disclosed system is also useful in performing protein interaction studies.
The disclosed two parts of the tag system are derived from bacterial ribose binding (RB) protein. In some embodiments, the disclosed ribose binding protein (RP-Tag, RPtag, Tag protein, Tag peptide, RPtag protein, RPtag peptide may be used to describe the presently disclosed proteins and peptides) is from the thermophilic bacterium Thermoanaerobacter tengcongensis (also referred to as C. subterraneous), and may be more stable than other RB proteins. However, other sources of RB proteins, for use with the disclosed RB-Tag system, are appropriate. In many embodiments, the disclosed ribose binding protein sequence may be altered/mutated to remove a putative N-terminal periplasmic localization sequence. In most embodiments, the disclosed RB-Tag sequences may also be altered to change naturally-occurring cysteine residues (Cys; for example, Cys 102) to serine residues (Ser; sequence of the intact protein below, Seq. 94).
The disclosed RB protein, from thermophilic bacteria, is very stable. In many cases, the disclosed RB protein has a melting temperature of over 100° C. The disclosed protein is also highly resistant to denaturants like guanidine hydrochloride and urea. Applicants have identified a peptide at the C-teminus of the RB protein that binds with very high affinity. Specifically, Applicants truncate the RB protein sequence at position 257, generating two RP-Tag fragments. The two fragments are referred to as RP-Tag Large (a.a. 1-257) and RP-Tag Small (a.a. 258-279; GEKIPNFIPAELKLITKENVQ; SEQ ID NO: 13). When expressed independently, the two fragments may be engineered to have short linker sequences at the C- and/or N-termini. The disclosed fragments may include any number of additional amino acids from the RB sequence (i.e. RP-Tag Large may comprise a.a. 1-260; and RP-Tag small may comprise a.a. 250-279), or amino acids from some other source, at the N- and/or C-termini. Additionally, the disclosed RP-Tag proteins may include fewer RB residues (i.e. RP-Tag Large may include a.a. 5-250, instead of a.a. 1-257).
Various embodiments of the disclosed proteins and peptides may include one or more changes selected from one or more of natural amino acid, synthetic amino acid, fusion, conjugation, derivatization, mutation, substitution, addition, or deletion. In many embodiments, the sequence of the disclosed RP-Tag proteins and peptides may possess less than 100% identity to the sequence of tte RB protein, for example less than 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50%, and greater than about 50%, 60%, 70%, 80%, 90%, or 95%. In some embodiments, the disclosed proteins and peptides may comprise one or more synthetic amino acids or residues.
The disclosed proteins and peptides may include one or more deletions. In some embodiments, the deletions may be truncations at one or both termini of the protein or peptide. In some embodiments, such deletions may aid in enhancing affinity or reducing affinity. The disclosed deletions may include from about 1 to about 20 contiguous, or non-contiguous residues, for example more than about 2 aa, 3 aa, 4 aa, 5 aa, 6 aa, 7 aa, 8 aa, 9 aa, 10 aa, 11 aa, 12 aa, 13 aa, 14 aa, 15 aa, 16 aa, 17 aa, 18 aa, or 19 aa, and less than about 20 aa, 19 aa, 18 aa, 17 aa, 16 aa, 15 aa, 14 aa, 13 aa, 12 aa, 11 aa, 10 aa, 9 aa, 8 aa, 7 aa, 6 aa, 5 aa, 4 aa, 3 aa, or 2 aa.
The disclosed proteins and peptides may have one or more amino acid changes in one or more functional and/or structural domains. For example, RPtag(small) peptide may include a domain that may aid in binding with another protein or peptide, such as RPtag(large), and another domain for stabilizing a bi-molecular complex (for example RPtag(large): RPtag(small)) or for stabilizing or destabilizing an intermediate form.
Binding Affinity
The disclosed RP-Tag proteins and peptides bind with specificity and with high affinity to each other. In many embodiments the equilibrium binding constant, Kd, is in the nanomolar range, for example less than about 100 nM, 10 nM, 1.0 nM, 0.1 nM, 0.01 nM. In many embodiments, the Kd is less than about 10 nM. As demonstrated below, in
Amino acid substitutions in the sequence of RPtag(small) peptide are useful in modulating the affinity for RPtag(large). In some embodiments, amino acid substitutions in the sequence of RPtag(large) peptide may be useful in modulating the affinity for RPtag(small). For example, amino acid substitutions at positions 2E, 18E, and 21Q may aid in increasing the affinity of RPtag(small) for RPtag(large). In some embodiments, the substitutions may be alanine, while in other embodiments enhancing mutations may be other than alanine, and at positions other than 2, 18, and 21.
Buffer Systems
Affinity and specificity may be changed depending upon the surrounding environment, for example the solution wherein binding occurs. In many embodiments, affinity may be affected by adding one or more organic solvents, alcohols, disulfide reducers, aromatics, sugars, salts, denaturants, detergents, etc. In some embodiments, the buffer system for the disclosed proteins and peptides may include one or more of DMSO, EtOH, MeOH, acetone, glycerol, BME, DTT, PG, imidazole, ribose, sorbitol, NaCl, KCl, NH4SO4, MgCl2, CaCl2, NiCl2, MnSO4, Gdn-HCl, urea, Tween20, TritonX-100, SDS. In some embodiments, salts may enhance or lessen binding affinity. In one embodiment, kosmotropic salts may aid in enhancing binding affinity, while chaotropic salts may decrease binding affinity. In many embodiments, NaCl and KCl may aid in stabilizing the interaction of RPtag(large) and RPtag(small). In these embodiments, the buffer may include a salt concentration of between about 5 mM and 5 M. In many embodiments, the effect on affinity may be similar for all peptides and protein, or may be different depending upon the sequence of the protein and/or peptide. In other embodiments, one or more compounds or molecules may be used to disrupt and/or lessen the disclosed interactions. In one embodiment, a pH buffer, denaturant, polyion, or imidazole may be used to disrupt binding. In these cases, the solution may help elute a target protein or target peptide from a solid support.
Disclosed herein are buffer systems for promoting and for disrupting interaction between the disclosed RPtag proteins. In some embodiments, buffers that promote binding may have pH between about 4 and 10, and a kosmotropic salt between about 10 mm and 5 M. In some embodiments, preferred buffers include about 0.1 M tris or phosphate pH 8.0, 3 M NaCl for binding. In some embodiments, buffers that may disrupt a RPtag complex may have a pH greater than about 10 and less than about 4, may comprise a chaotropic salt, may comprise imidazole, and combinations thereof. In some embodiments, preferred buffers include about 0.1 M tris or phosphate pH 8.0, 3 M imidazole for elution.
Protein Expression
The large RP-Tag protein is also useful in aiding the stability and expression of other protein sequences to which it is fused. In many embodiments, fusion proteins, having the sequence of the Large RP-Tag protein may express to greater than about 400 mg/L when expressed in bacteria (for example BL21(DE3) E. coli). In some embodiments, high expression of stable, functional, fusion proteins may be achieved with pH-stat fed-batch bioreactor and methods of using the stated bioreactors.
The disclosed RP-Tag proteins may be expressed in or from a variety of prokaryotic and eukaryotic cell and systems. In some embodiments, the RP-Tag protein is expressed from a yeast cell, bacterial cell, mammalian cell, insect cell, plant cell, etc., such as Saccharomyces cerevisiae, Pichia pastoris, Human Embryonic Kidney cell, Chinese Hamster Ovary Cell, Spodoptera frugiperda, etc. or extracts thereof. In some embodiments, the disclosed proteins and peptides may be chemically synthesized.
The disclosed RP-Tag interaction may be stabilized in the presence of ribose. Ribose is bound by the large RP-Tag protein, and its interaction with RP-Tag Large may help to stabilize the structure of this fragment and may also help to stabilize interaction between the two RP-Tag fragments.
Solid Supports
The disclosed Tag proteins may be affixed to a solid support to aid in isolating the complement Tag protein. For example, in some embodiments, the Large RP-Tag protein may be affixed to a matrix for a column, and a fusion protein comprising the Small RP-Tag protein may be combined with the matrix (either in solution [or batch processing], or by adding the RP-Tag fusion protein to a column comprising the solid matrix/RP-Tag protein, as in Example 1, below) to isolate and purify the fusion protein. In other embodiments, the Small Tag protein is affixed to the column matrix to aid in binding a fusion protein comprising the Large Tag protein. Thus, a target protein may be fused to either the Small or Large Tag protein, and may be fused to either the C- or N-terminus of either protein. In some embodiments, the fusion protein may include a linker sequence between the Tag sequence and that target protein sequence. In many embodiments, this linker sequence may be from about 1 a.a. to about 30 a.a. in length. In some embodiments, this linker sequence may add functionality to the fusion protein, for example by introducing a labelling sequence, cleavage sequence, or recognition sequence.
Suitable resins for immobilization may comprise a bead of polymeric matrix (for example but not exclusive to: agarose, Sepharose, dextrans, acrylamide, bisacrylamide, silica, methacrylate, and various mixtures and cross linking formulations thereof), along with a chemistry for coupling to the peptide or protein (e.g. an aldehyde, maleimide, N-Hydroxysuccinimidyl ester, halo-acetyl group, sulfhydryl (activated or free), hydrazide, hydrazine, amine, alkyne, azide, carboxyl group, or other moiety commonly known in the art), that may or may not be on the end of a spacer which is attached to the polymer matrix.
A variety of methods may be used to affix an RP-Tag protein to a solid support. In some embodiments, it may be useful to add one or more amino acids to the RPtag protein to aid in linking the RPtag protein to the solid support. In other embodiments, the linkage may be chemical, for example via cysteine, di-sulfide bond, primary amines, amide bonds, or other covalent chemistry. In one embodiment, a Cys residue may be engineered in the RPtag protein to allow the protein to link a solid support via a thioether bond (e.g. using SULFOLINK™ technology from ThermoFisher Scientific). By another method, the RPtag protein or peptide might be immobilized via free amine groups to aldehyde resin, thus forming an imine, and then reduced via sodium cyanoborohydride to form a stable secondary amine.
Modifications—Tags, Linkers, Reporters, Etc.
The disclosed RPtag proteins may be labeled to aid in visualizing or locating one or both proteins. Suitable label and methods of labeling proteins are well known in the art. In some embodiments, specific amino acid residues may be targeted for attaching one or more labels. In other embodiments, target sequences (for example the linker sequences described above) may be added to the RPtag proteins to facilitate labeling. In some embodiments the label is visible (e.g. dyes or fluorescent labels), or the label may be visualized with detector equipment (e.g. radioactive labels, fluorophore, radioactive isotopes, chromophores, metals for electron microscopy like gold and iron, quantum dots, etc.), or other labeling techniques well known to those skilled in the art. In one embodiment, the RPtag protein is labeled with rhodamine.
Mutations may be introduced in the Tag protein using a variety of methods well known to those of skill in the art. In some embodiments, as discussed above, additional amino acids may be added to the Tag protein sequence to create linker sequences that may be useful in adding a label, tag, or other adduct to the protein. In other embodiments, the amino acid sequence of the Tag protein may be mutated to change one or more amino acid residues. In these embodiments, it may be useful to create specific amino acid substitutions to help increase or decrease affinity between the two Tag proteins. As one example, a Small mutant Tag protein may be engineered to have greater affinity for the Large Tag protein to aid in displacing, or competing away the disclosed Small Tag protein. In other embodiments, amino acid mutations may help to lower the affinity of the Large Tag protein for the Small Tag protein.
Protein Stability
The disclosed Tag protein affinity system is resistant to conditions that normally disrupt protein-protein interactions. Typically, protein-protein interactions are sensitive to disruption by changes in pH, ion concentrations, temperature, and denaturant concentration. For example, typical protein-protein concentrations may be disrupted by increasing or decreasing the pH of a solution containing a protein-protein interaction above about 8.0 pH or below about 6.5 pH. In many embodiments, the disclosed protein-protein interaction is stable in pH above 8.0 pH and below 6.5 pH. In some embodiments, the disclosed interaction is stable in high concentrations of one or more denaturant compounds (e.g. urea, guanidine, etc.), wherein the concentration of denaturant is greater than about 1M.
“Polypeptide,” “protein,” and “peptide” are used interchangeably to refer to or describe a linear or branched chain of amino acid monomers linked by peptide bonds. Individual positions within those chains may be referred to as a “residue,” or “amino acid.” The disclosed polypeptides, proteins, and peptides may be of any length and comprise any number of natural or synthetic amino acids.
“Homology,” “homologous,” “identity,” “identical,” “similar,” and “similarity” as used herein refer to a degree of nucleic acid and/or amino acid sequence similarity between two optimally aligned nucleic acid or peptide molecules. Percent homology and identity are determined by comparing positions in two or more sequences, aligned for purposes of such a comparison. In many cases, one of skill in the art can use one or more computer applications to determine such values, for example BLAST. Comparing equivalent positions in different sequences may identify the same residue or nucleotide—this is referred to as identity. In contrast, were the equivalent positions have amino acid residues with similar characteristics or properties (e.g. size, polarity, charge, etc.) the amino acids may be homologous but not identical.
Non-covalent interactions refer to interactions based on non-covalent forces, such as ionic, hydrophobic and hydrogen bond-based interactions. Non-covalent interactions do not include interactions based upon two atoms sharing electrons.
Affinity may be expressed in terms of the equilibrium binding constant Ka, or dissociation constant, Kd or KD. Kd is expressed as a concentration and can be determined by measuring the association rate constant, ka, and dissociation rate constant, kd, and determining their ratio (kd/ka). One of skill in the art is readily able to determine affinities using a variety of techniques and methods. Typically, one of skill in the art may determine an equilibrium constant or Kd, by varying input concentrations of one component (here, [RP-Tag Large] or [RP-Tag Small]) to achieve equilibrium, and measuring the relative concentration of the complex (here [RP-Tag Large:RP-Tag Small]). Other techniques are able to monitor such interactions in real-time to determine on-rates and off-rates.
One embodiment of the disclosed RP-Tag system was tested by creating a column with one component bound to a solid, agarose-based matrix. In these experiments, a SulfoLink™ Immobilization kit (ThermoFisher scientific) was used to affix RP-Tag Large to a solid support, according to the manufacturer's instructions. For these experiments, an N-terminal linker was added to RP-Large that included a Cys residue. One of skill in the art is able to select various techniques and chemistries to aid in affixing either RP-Tag protein to a solid support matrix.
The amount of protein linked to the column was determined using a BCA assay with Bovine Serum Albumin (BSA) as a standard. This showed that 4.5 mg RP-Tag Large was immobilized onto 2 mL of the SulfoLink™ resin to create a RP-Tag Large-linked resin. The linked resin was poured into an included column and the column capped with a frit included in the kit (see photos in
Methods
An E. coli codon-optimized gene encoding tteRP-Tag Large was synthesized using solid-state methods. This gene was then cloned into a pET-28a(+) expression vector.
Labeled and unlabeled RP-Tag Small proteins were synthesized solid state, resuspended in DMSO (1-10 mM final peptide concentration) and stored at −20° C. until needed. Prior to use, the proteins were thawed and diluted into an appropriate buffer.
RP-Tag Large Expression and Purification
Chemically competent BL21(DE3) E. coli were transformed with 50 ng expression plasmid, streaked onto Luria Broth (LB)+50 mg/L kanamycin agar plates and grown at 37° C. overnight. Single colonies were then picked and grown in Fernbach flasks in LB+50 mg/L kanamycin at 37° C. with continuous shaking at 225 RPM until OD600=0.6. The temperature was then dropped to 25° C. and the cultures induced with 20 mg/L Isopropyl β-D-1-thiogalactopyranoside (IPTG) and grown for an additional 18 h.
Cultures were submitted to centrifugation to pellet bacterial cells. Supernatant was discarded and cell pellets resuspended in 20 mM sodium phosphate pH 8.0, 300 mM NaCl, 10 mM 2-mercaptoethanol, and 10 mM imidazole. Cells were lysed enzymatically (Lysozyme, DNAaseI, 5 mM MgSO4 1 hour on ice), cell debris pelleted by centrifugation, and the clarified supernatant loaded onto a NiNTA column equilibrated with the lysis buffer. Protein was then eluted with a step gradient of imidazole (10 mM-250 mM), and protein-containing fractions pooled and dialyzed against 20 mM sodium phosphate 8.0, 150 mM NaCl, and 10 mM 2-mercaptoethanol.
Dialyzed protein samples were flash frozen in liquid nitrogen and stored at −80° C. until use. Concentrations were determined either using a BCA assay using Bovine Serum Albumin as a standard, or by A280 nm using a calculated ε280=4,470 M−1 cm−1. For pH-stated fed-batch bioreactor protocols, an identical protocol was used except cultures were grown in a 10-L New Brunswick Bioreactor, LB was additionally supplemented with 20 g/L glucose and 0.6 g/L magnesium sulfate, and pH was maintained between 6.85 and 6.95, adding 50% glucose and 1.5% MgSO4 mixture if the pH increased over 6.95 by peristaltic feed pump, and 30% ammonium hydroxide if pH dropped below 6.85 by peristaltic feed pump. Fed-batch cultures were induced at OD600=6 with 1 mM IPTG. Purified protein was >95% pure as judged by SDS-PAGE stained with coomassie brilliant blue R-250.
50 nM of RP-Tag Small (see Sequence 3, below) with an N-terminal Rhodamine B label was incubated with increasing concentrations of either RP-Tag Large (see Sequence 2) or Bovine Serum Albumin (BSA) in 50 mM Tris pH 8.0, 150 mM NaCl, 10 mM 2-mercaptoethanol, and 0.005% Tween 20 for 5 min in black 96-well plates at room temperature.
Anisotropy was then measured, and a Kd calculated by fitting the data to the equation f=y0+(ymax−y0)*(Ptot+x+Kd−sqrt((Ptot+x+Kd){circumflex over ( )}2−4*Ptot*x))/(2*Ptot), where y0 is the baseline anisotropy, ymax is the maximum anisotropy, Ptot is the fixed concentration of labeled peptide used, x is the variable concentration of protein used, and Kd is the measured Kd. Fitting of the data resulted in a calculated Kd of 8 nM for this interaction, and detected no binding to BSA (see
Binding of RP-Tag Small to Immobilized RP-Tag Large
25 mL of 1 μM Rhodamine-6B labeled RP-Tag Small, in 50 mM Tris pH 8.5, 150 mM NaCl, 5 mM EDTA, was flowed over the column of immobilized RP-Tag Large protein. RP-Tag Small bound the column and formed a visibly bright red band (rhodamine B) at the top of the resin (see
Table 1 summarizes results from elution tests using a variety of conditions. Briefly, after binding, the RP-Tag Small red band was not observed to appreciably diffuse and/or elute after washing the column (and band) with various solutions. For these tests, 10 mL (5 column volumes) of various buffers were added to the column. The tested buffers ranged from about pH 1.5-13.7 and about 1 M sodium hydroxide. These results (see
Table 1. Summary of elution trial data. The test system used was a column equilibrated with 50 mM Tris pH 8.5, 150 mM NaCl, 5 mM EDTA, and applied 25 mL 1 μM Rhodamine-6B labeled RP-small peptide in the same buffer. In cases where D-ribose was used, the indicated concentration was also included in the equilibration and loading buffer.
Diffusion and/or elution of the rhodamine-labelled band required subjecting the column to very strong buffers. For example, elution was seen with a buffer comprising 100 mM glycine and pH 1.5, as well as a buffer comprising 0.1 M Tris pH 7.5+6 M Guanidine-HCl. Even after elution with these strong buffers, the column was able to be re-equilibrated with neutral buffer (specifically 50 mM Tris pH 7.5), and its ability to bind RP-Tag Small was restored. These results indicate that the RP-Tag resin can be effectively washed with high concentrations of hydroxide (e.g. 1 M sodium hydroxide), low pH buffer (e.g. 100 mM glycine pH 1.5) and high concentrations of denaturants (e.g. 6 M guanidine hydrochloride), and still be regenerated to a functional state.
Conditions were investigated in which the RP-Tag Large resin would bind RP-Tag Small proteins under strongly denaturing conditions (e.g. 6 M Gdn-HCl). RP-Tag Large's ability to bind ribose was investigated. RP-Tag Small was bound as described above with 10 mM, 100 mM, and 1 M D-ribose. All concentrations of D-ribose significantly slowed diffusion of the bound rhodamine band with 5 column volumes (CVs) washing. About 100 mM D-ribose stopped virtually all peptide elution from the column, while at 1 M D-ribose even diffusion within the column was reduced to modest levels.
It should be noted that no tested concentration of D-ribose was able to stop diffusion of the rhodamine band entirely. In 6 M guanidine the capacity of the column is likely reduced and extensive washing would almost certainly cause the target to leach to some extent. Nonetheless, these results indicate that the inclusion of increasing concentrations of D-ribose can stabilize RP-tag under denaturing conditions, and make it an effective purification tool even with high concentrations of denaturants.
Conditions under which the labeled RP-Tag Small could be eluted at neutral pH were investigated. These experiments were directed to eluting bound RP-Tag Small using unlabeled RP-Tag Small—that is, disrupting the complex by competition. For these experiments, 100 μM of unlabeled RP-Tag Small in 0.1 mM Tris pH 7.5 was used. Slight diffusion of the rhodamine band within the column, was observed. In addition, these competition experiments successfully eluted a small amount of labeled RP-Tag (small) protein from the column.
These results demonstrate that bound RP-Tag proteins may be competed off the column under neutral conditions. In some embodiments, higher affinity RP-Tag proteins may be engineered to help compete with one or more of the existing RP-Tag proteins. In some embodiments, the affinity of the interaction may be modulated by mutating one or more residues to raise or lower the interaction's strength/affinity. In some embodiments, a closely related protein or peptide may be used for completion and/or a multimeric peptide used.
Equilibrium binding affinities (Kd) of the native RPtag(large)/RPtag(small) interaction was compared to a commonly used, commercially available epitope tag antibody and its corresponding tag by fluorescence anisotropy (mouse monoclonal antibody purchased from ThermoFisher Scientific (4E3D10H2/E3)). In these studies, the tag sequence was GHHHHHH (SEQ ID NO: 1) with an N-terminal rhodamine B.
The indicated concentrations of RPtag (large) and an anti-His tag antibody (4E3D10H2/E3 purchased from ThermoFisher Scientific) were incubated with 1 nM rhodamine labeled native RPtag (small) peptide and 6×His peptide (Rhodamine-GHHHHHH), respectively, and fluorescence anisotropy measured. BSA incubated with native rhodamine labeled RPtag(small) is included as a control for non-specific binding. Kd's were calculated according to the equation f=y0+(ymax−y0)*(Ptot+x+Kd−sqrt((Ptot+x+Kd){circumflex over ( )}2−4*Ptot*x))/(2*Ptot), where y0 is the baseline anisotropy, ymax is the maximum anisotropy, Ptot is the fixed concentration of labeled peptide used, x is the variable concentration of protein used, and Kd is the measured Kd. We measured a Kd of 0.2±0.1 nM for RPtag(large) binding RPtag(small), and a Kd of 6±1 nM for the antibody/6×His tag pair. There was no detectable binding of RPtag (small) to BSA up to the indicated concentrations.
Results presented in
The ability of the disclosed proteins and peptides to function after being subjected to thermal stress was also tested. Here again the RPtag (large) and anti-6×His antibody were selected for analysis. Briefly, the RPtag(large) or the anti-6×His antibody was subjected to sequential rounds of boiling and recovery. Specifically, the proteins were subjected to sequential rounds of: 5 min boiling in buffer, followed by recovery for 1 min on ice. After each round of boiling/recovery, the proteins were assayed for binding to their corresponding epitopes and the results plotted.
RPtag (large) and an the anti-His tag antibody (4E3D10H2/E3 purchased from ThermoFisher Scientific) were placed in a solution at about 0.1-1 μM [final] in a buffer of 50 mM Tris pH 8.0, 0.005% Tween20. The protein solutions were repeatedly heated for 5 min at 95° C. and then recovered on ice for 1 min. After each round of heating/cooling, an aliquot was taken and diluted to 100 nM, and incubated with 100 nM either rhodamine labeled RPtag (small) or rhodamine labeled 6x-His peptide (sequence: Rhodamine-GHHHHHH), and the fluorescence anisotropy measured. Fraction binding was calculated via the equation F=(r−rmin)/(r0−rmin) where F is the fraction binding, r is the measured anisotropy, rmin is the anisotropy in the absence of binding protein, and r0 is the anisotropy before any boiling trials.
As shown in
A second stress test was performed on the proteins by subjecting them to a 15 min 121° C. autoclave cycle, after which the proteins' function was assayed.
Specifically, RP-tag (large) and the anti-6×His antibody (4E3D10H2/E3 purchased from ThermoFisher Scientific) at 5 μM were subjected to a 15 min 121° C. autoclave cycle with slow exhaust to prevent boiling (total time>100° C.˜60 min) in 50 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA, 10 mM β-ME, and the Kd measured as above.
As expected, the antibody was completely destroyed by this treatment, losing all detectable binding to its target peptide (see
To examine the efficacy and specificity of the disclosed proteins, peptides, systems, and methods, fusion proteins were created comprising the disclosed proteins and peptides, and several biomolecules of interest. In one example, RPtag (large) or RPtag (small) were conjugated to a resin of agarose beads. In these experiments, the RPtag sequences were engineered to include N-terminal cysteines, which could be used to covalently bind activated agarose (via the manufacturer's instructions; SulfoLink™ resin purchased from ThermoFisher scientific). Immobilization efficiencies were about 2 mg RPtag (small)/mL resin, and ˜38 mg RPtag(large)/mL resin).
Protein Purification
Briefly, codon-optimized DNA coding sequence of each protein was synthesized solid state and then sub-cloned into the pET-28a(+) bacterial expression plasmid. Chemically competent BL21 (DE3) were transformed with the expression plasmids and grown on LB agar+50 μg/mL kanamycin sulfate at 37° C. overnight. Colonies were picked and grown in shaker flasks in LB+50 μg/mL kanamycin sulfate (200 RPM) at 37° C. until OD600=0.6. The temperature was then dropped to 25° C. and expression induced with 1 mM isopropyl μ-D-1-thiogalactopyranoside for 16 hrs. Cells were then harvested by centrifugation and lysed enzymatically with lysozyme and DNAase in 20 mM Tris pH 8.0, 300 mM NaCl, 10 mM imidazole, 5 mM MgCl2. Cell debris was pelleted by centrifugation, and proteins purified by single-step NiNTA chromatography (10 mM-500 mM imidazole step gradient). Concentrations were determined by absorbance using ε595=100,000 M−1cm−1.
Column Production
SulfoLink™ resin was purchased from ThermoFisher Scientific. For immobilization of RPtag (large), 200 mg protein/mL resin was incubated at room temperature for 1 hour in Tris pH 8.5, 1 mM EDTA. The RPtag(large)-resin was then washed and incubated in the same buffer+10 mM cysteine. Next, the RPtag(large)-resin was packed into 1 mL FPLC-columns (Gold Biotechnology, Inc., St. Louis, Mo.; see
Binding Experiments
After purification of the tagged proteins, each was applied to its complementary column to evaluate binding.
In some embodiments, immobilization of the RP-tag(small) peptide may allow for the use of a high-solubility, expression and solubility enhancing tag on either terminus of the protein of interest. In other embodiments, immobilization of the RP-tag(large) protein may allow for the use of a small, minimally perturbing tag on the protein of interest, again at either terminus.
As a test for specificity, non-complementary proteins were applied to each column using the same procedure described above. In these experiments, only a small amount of non-specific binding was observed. This background binding is not uncommon and may, in some cases be expected with agarose-based chromatography resins. In some cases, color in the photographs was enhanced to aid in visualization. Where such enhancement was performed, each panel received identical enhancements.
Next, N-terminal and C-terminal fusions of both RPtag (large) and RPtag(small) with a red fluorescent protein (tagRFP) were constructed. TagRFP allows visualization of the proteins (proteins also had an 8×his tag on the opposite terminus to aid in rapid purification). After purification of the tagged proteins, each was applied, separately to its complementary column to evaluate binding.
Briefly, columns were equilibrated in 20 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA (TEN buffer), then 25 mL of 1 μM tagged protein in TEN buffer was applied to the column at a flow rate of 1 ml/min. Thereafter, the column was washed with 10 mL of TEN buffer, and the results recorded by photograph (
To define the mechanism of RPtag (large) and (small) binding, the reaction order of the rate-limiting step was determined.
Results
For these experiments, 1000 nM RPtag(large) was incubated with increasing concentrations of native RPtag (small) (from about 0.6 to 10 nM). These experiments identified a linear increase in the initial rate of formation of the complex (RPtag(large):RPtag(small)). This linear increase indicated that the rate limiting step is first order with respect to RPtag(small) (
Panel a depicts representative association rate kinetics traces varying native RPtag small. Rhodamine labeled RPtag small at the indicated concentration was incubated with unlabeled RPtag large and the association measured by fluorescence anisotropy. L-S complex concentration was calculated by the equation (r−rmin)/(rmax−rmin)*[S] where r is the measured anisotropy, rmin is the anisotropy in the absence of any RPtag large, rmax is the anisotropy measured in the presence of at saturating RPtag large, and [S] is the total concentration of RPtag small used in the experiment. As shown in
Panel c shows initial velocities of traces represented in panels a (black) and b (red). The first 5 min of data were fit with a line, and plotted as a function of concentration. S(total) is plotted as a function of the total concentration of RPtag small used in the reaction, S (corrected) is plotted as a function of the concentration of S corrected for Keq as detailed herein. The slope of the line using S (total) is 0.035 min−1, in much worse agreement with the data and single exponential fit rate constant than the 0.091 min−1 slope of the S(corrected) line. Data shown are mean±SE (n=3).
Next, 1000 nM RPtag (small) peptide was incubated with increasing concentrations of RPtag (large) protein (from about 0 to 10,000 nM). These experiments resulted in no change in the rate (except at 0 nM RPtag(large)). These results indicated that the reaction is 0th order with respect to RPtag (large) (
Panel b presents the representative kinetics traces varying native RPtag large. Indicated concentrations of RPtag large were incubated with 1000 nM RPtag small and anisotropy measured. All calculations were as in panel a.
Taken together, these studies demonstrated that the rate liming step of complex (RPtag(large):RPtag(small)) formation is first order only with respect to RPtag (small), consistent with a unimolecular process. Without wishing to be limited, this suggests the possibility of a conformational change in the RPtag(small) peptide prior to binding. The conformational change being from a “non-binding” (S) to “binding” (S*) state. This conformation change is then followed by a much faster bi-molecular binding event (to RPtag(large)). Again, without wishing to be limited, the data suggest a missing amplitude in the binding kinetics. Specifically, this may represent the proportion of the binding reaction that occurred in the dead time of the instrument (˜1 min), and therefore the amount of RPtag (small) that was already in the S*, binding state at the start of the experiment. From the missing amplitude and total amplitude, an equilibrium constant for S and S* can be calculated (Keq˜1.6).
These values can then be used to correct for the true concentration of S in the initial rate plots (
Knowing the Keq and kf (forward rate constant) for the reaction, the reverse rate constant (kr) of 0.056 min−1 was calculated.
Resulting kinetics observed in these studies are shown in
Fitting to a line yields a rate constant (koff)=0.0091 min−1 (
These rate constants were used to simulate the mechanism shown in
Mutagenesis studies were carried out on the native RPtag(small) peptide sequence to attempt to identify the region required for binding to RPtag(large), and to identify specific mutations responsible for improving binding affinity or improving binding kinetics. Additionally, these studies might identify a minimal binding domain in RPtag(small) leading to a decrease in the size/number of amino acids in the RPtag(small) peptide.
Effect on Binding Equilibrium
First alanine scanning mutagenesis was performed along the sequence of RPtag(small). Specifically, each amino acid position in RPtag(small) was changed to alanine and the Kd of the resulting peptide measured. The Kds of these alanine mutants is shown in Table 2. These studies identified a cluster of amino acids from about F7 to about K17 that, when changed to alanine, significantly impaired binding affinity. This indicated that the RPtag(large) binding region lies within these about 11 amino acids. Structurally, this region corresponds roughly to the β-sheets on the crystal structure of the tteRBP (PDB 2IOY). Interestingly, despite being in the middle of the β-sheet region, P9 did not impair affinity when changed to alanine. Surprisingly, 3 mutations significantly increased binding affinity to RPtag(large)—E2A, E18A, and Q21A. These positions all lie outside of the putative binding region, and should therefore be amenable to incorporation into the sequences without disrupting the necessary interactions for binding.
Next, sequential truncation mutagenesis from both the N- and C-termini of the peptide was performed (see Table 2). Beginning at about position Nd7 (deletion of 7 amino acids from N-terminus) impairments of ˜10 fold or greater were identified. This level of Kd reduction was also seen with Cd5 mutations (removal of 5 amino acids from C-terminus). These truncations, respectively, correspond to the F7 and K17 identified above in the alanine scanning studies.
A second region of the irregular β-sheets was found between about L12 and K17. This region appears to also be involved in binding the RPtag(large) protein, like the first identified region between about F7 and E11. In this second region, Kd impairments of >1000-fold resulted from truncating into the region from either the N or C terminus. Surprisingly, Nd8 (removing the first 8 amino acid positions while leaving P9 as the N-terminal amino acid) resulted in improving the binding affinity for RPtag(large), relative to the truncations to positions 7 or 9. This data aligns well with the observation that mutation P9A did not significantly impair the binding affinity, but A mutations at positions 8 and 11 did.
Effect of Binding Rates
The library of mutant RPtag(small) peptides, described above, was next analyzed to measure off rates (koff). The on rate was also calculated from measured koff and Kd (S*-LS transition), except instead of using unlabeled native peptide as the competitor, we used unlabeled Nd2,P5A,E18A (the tightest binding RPtag(small) peptide identified). These studies showed that kon and koff roughly tracked with Kd. However, there were notable exceptions. For example, although mutation P5A (proline at position 5 of RPtag(small) changed to alanine) did not have a significant effect on Kd, the mutation increased the kon and koff by a factor of ˜7. This indicates, without wishing to be limited, that P5 may constrain the structure of the peptide. The increased flexibility imparted to the peptide by the P5A mutation may decrease the energy barrier required for binding/unbinding. N6A had the opposite effect, decreasing the on and off rates without having a significant effect on Kd, indicating that this mutation may increases the energy barrier.
Forward and reverse rate constants for the S-S* transition revealed additional notable mutants. Measured Keq's and kf's as well as the calculated kr's for the alanine scanning and truncation library peptides were all similar, with the exception of K3A, K13A, K17A, Nd3 truncations and further, and Cd5 truncations and further. A Keq or kf (and correspondingly kr) could not be determined for these mutants as the association reaction appeared to be completed within the dead time of the instrument (˜1 min). Of note, the Nd3 deletion corresponds to deletion through K3, and Cd5 corresponds to deletion through K17. This indicated that lysines in the peptide may play a significant role in limiting the rate of LS complex formation. Moreover, again without wishing to be limited, the rate limiting structure/interaction may be alleviated by the mutation or removal of either lysine.
The kinetics data described above has at least two possible interpretations. Either these mutations cause a substantial increase in the rate of the S-S* transition, or they shift the Keq of S-S* such that it heavily favors S* (or some combination of the two). Without wishing to be limited, the end result may be a substantial increase in the rate of formation of the LS complex. Of the 3 lysines, only K3 falls outside the proposed RPtag(large)-binding region, which may allow the making mutations at or truncations of K3 much more readily than at either K12 or K17.
Mutant RPtag(Small) Peptides with One or More Alanine Substitutions and/or Truncations
Several of the mutations identified above, in the alanine scanning mutagenesis and truncation mutagenesis, were combined and analyzed. These experiments were intended to potentially identify two peptides: the smallest peptide with antibody-like binding affinity to RPtag (large) (Kd≤10−8 M) and fastest kinetics, and the peptide with the tightest binding regardless of size or kinetics.
These studies identified the Nd8 truncation with the Cd4 truncation (Nd8Cd4) as having favorable characteristics in terms of size, kinetics, and Kd. This peptide has a size of 9 amino acids, a Kd of ˜14 nM, and binding/unbinding kinetics completed within the dead time (see Table 1; apparently meeting the criteria of the first desired peptide). Next, Nd2, p5a, and e18a were combined and analyzed. This peptide possessed a Kd of ˜47 pM, which appeared to meet the criteria for the second desired peptides.
These two identified RPtag(small) mutant peptide sequences were subjected to additional testing, described below. Their sequences are:
The Kds of Nd8,Cd4 for RPtag(large) was measured in the presence of buffers of different pHs ranging from 1.5 to 13. These studies found that native, Nd8Cd4, and Nd2p5ae18a all showed good stability over a wide pH range, and possessing a maximum affinity between pH 4-10, with a relative maximum at ˜pH 8 (
For these experiments, Kds were measured by fluorescence anisotropy as described above in the following buffers, all at 100 mM with 0.005% Tween 20: glycine pH 1.5, glycine pH 2.0, glycine pH 3.0, acetate pH 4.5, 2-(N-morpholino)ethanesulfonic acid pH 6.0, tris pH 7.0, tris pH 7.5, tris pH 8.0, tris pH 8.5, borate pH 10.0, phosphate pH 11.5, phosphate pH 13.0.
Reagent Screening
To determine reagent compatibility with the disclosed system, an additive screen was performed, wherein the Kd between RPtag (large) and 3 RPtag (small) peptides—native, Nd8Cd4, and Nd2p5ae18a—were tested in the presence of several common buffer additives (Table 3).
These experiments demonstrated that the disclosed proteins, peptides, and systems were robust in the presence of a number of organic compounds (DMSO, methanol, ethanol, glycerol, and acetone), reducing agents (DTT), and detergents (Tween20 and TritonX-100). Interestingly, kosmotropic salts such as NaCl and KCl significantly stabilized the interaction between RPtag(large) and RPtag(small). In particular, the RPtag(small) mutant peptide Nd8Cd4 showed an increased affinity of about >100-fold. In contrast, addition of chaotropic salts, such as MgCl2 and CaCl2, resulted in a destabilized LS complex. Surprisingly, addition of imidazole significantly destabilized the interaction of RPtag(small) and RPtag(large). Under the conditions of this experiment all apparent binding affinity between the two RPtags was removed.
Next the affinity of RPtag(small) mutant peptide Nd8Cd4 was analyzed as a function of NaCl and imidazole. These studies identified a dose-dependent enhancement of affinity in the presence of NaCl, and impairment of affinity with imidazole (not shown).
Because of the observed stabilizing character of NaCl and destabilizing character of imidazole on Nd8Cd4 binding, these reagents, among others, were incorporated into a novel buffering system for use with the disclosed proteins, peptides, systems, and methods. In particular, the effect of this novel buffer on binding of RPtag (small) Nd8Cd4 to an immobilized RPtag(large) column was studied. These experiments included eluting both (1) rhodamine labeled peptide and (2) N-terminally RPtag(small) Nd8Cd4 tagged with tagRFP from a column under native conditions. Generally, native conditions is taken to mean at or near biological conditions under which many proteins are folded, e.g. pH 4-9 at moderate temperature (4° C.-30° C.) in the absence of denaturants (e.g. guanidine hydrochloride, urea, SDS). This is in contrast to denaturing conditions, which generally rely on extreme pH (e.g. ≤2) denaturants, or high temperature to unfold proteins.
Briefly, columns, as described above, were first equilibrated with 10 mL 100 mM Tris pH 8.0, 3 M NaCl, 0.005% Tween20. Next, 1 mL of 10 uM rhodamine-tagged Nd8Cd4 or tagRFP containing an N-terminal RPtag (small) Nd8Cd4 tag was applied in the same buffer. The column was then washed with 10 mL of the same buffer. Finally, bound target molecules were eluted with 10 mL of 100 mM Tris pH 8.0, 3 M imidazole, 0.005% Tween20.
These experiments demonstrated that both the protein and the peptide could be eluted as a tight band. This demonstrated the efficacy of the disclosed buffering system for use in binding and elution of peptides and proteins at neutral pH. These same conditions caused native and Nd2p5ae18a peptides to bind in a tight band. Although these peptides were able to be eluted, instead of eluting as a tight band, the eluted band spread through the resin, creating a diffuse band. This suggested that modification of the disclosed buffering system for these sequences may be beneficial.
The disclosed proteins and peptides were used in precipitation/pull-down assays. Specifically, rhodamine was tagged with the RPtag(small) Nd8Cd4 peptide and RPtag(large) was immobilized on a resin, as described above.
Briefly, 100 μL 1000 nM rhodamine labeled RPtag(small) peptide was incubated with 2.5 uL wet settled RPtag (large) resin generated as (as described above) in phosphate buffered saline (PBS; Gibco)+1% BSA+0.005% Tween 20 at 4° C. for 60 min with continuous orbital shaking. Samples were then washed 3× with 500 μL of the same buffer with no peptide to remove unbound peptide. Bound RPtag(small) peptides were then eluted with 100 μL 100 mM glycine pH 1.5+0.005% Tween20, and the pH raised by adding 1 μL 1 M borate pH 10.0. The amount eluted was calculated from the blank subtracted fluorescence of the elution versus the load. After each elution, resin was washed once with 500 μL 6M GdnHCl and once with 500 μL phosphate buffered saline (Gibco)+1% BSA+0.005% Tween 20, before repeating the assay. Data shown in
Sequential precipitation/pulldown trials were performed with the rhodamine labelled RPtag(small) Nd8Cd4 peptide, and the RPtag(large)-resin. After binding and pull-down, the rhodamine-peptide was eluted in 100 mM glycine pH 1.5, the column was washed with water and 6 M guanidine hydrochloride, and then re-equilibrated with buffer (50 mM Tris pH 8.0, 10 mM EDTA). RPtag(small) Cd12 peptide as used as a negative control due to its similar size (9 amino acids) and markedly lower binding affinity for RPtag(large).
The disclosed proteins and peptides were used for detection assays. In these experiments, RPtag (small) was recombinantly fused to an alkaline phosphatase. Specifically, the RPtag(small) peptide Nd8Cd4 was fused to the N-terminus of a highly active monomeric alkaline phosphatase (PhoX class) from Pasteurella multocida. RPtag (large) with an engineered cysteine was immobilized to a white, polystyrene 96-well plate via activated maleimide (ThermoFisher) according to the manufacturer's instructions. Control wells were blocked with cysteine. 100 μL of 12.4 μM RPtag(small) Nd8Cd4-labeled alkaline phosphatase was added to both the experimental and control wells and then incubated for 1.5 hrs at room temperature. Wells were washed 3× in 50 mM Tris pH 8.0, 150 mM NaCl, 1% BSA, 0.05% Tween 20, and then either assayed with 0.25 mM CSPD purchased from ThermoFisher (a luminescent alkaline phosphatase activity probe) and measured immediately at room temperature, or 2 mM p-nitrophenyl phosphate (an absorbance alkaline phosphatase reagent) in 50 mM Tris pH 9.5, 150 mM NaCl, 100 μM CaCl2), 0.2% Tween 20) and measured after 60 minutes at room temperature. Measurements were taken on a SpectraMax i3 (Molecular Devices). For luminescence, the plate was placed into the instrument, mixed for 60 seconds in the dark, and then all wavelengths were collected from each well. For absorbance, after 60 minutes the samples were transferred to a transparent 96 well plate and the absorbance measured at 405 nm.
As shown in
Additional mutagenesis studies were carried out on the RPtag(large) and RPtag(small) sequences to further study binding. First, the ability to engineer compensatory mutations in each protein was investigated. Second, native RPtag(large) peptide sequence was modified to study this protein's ability for expanded recognition/specificity.
Compensatory Mutations in RPtag(Large) and RPtag(Small)
11 PDB crystal structures of periplasmic sugar-binding proteins, from diverse species, were aligned to further investigate residues involved in binding specificity (
Of the 11 RPtag(small) PDB crystal structures investigated, 8 sequences showed strong conservation of leucine (L) or valine (V) at position 14. 7 of the 11 RPtag(large) PDB crystal structures, possess an aromatic residue at position 137.
The possibility of creating compensatory mutations in the two sequences was investigated by modifying position 14 of RPtag(small) and position 137 of RPtag(large). First, a positively charged lysine (K) was introduced at position L14 of RPtag(small) (
These experiments demonstrate that applicants have identified the pocket/cleft in the RPtag (large) protein useful for interacting with the RPtag(small) peptide. Further, as one of skill in the art would understand, additional positions, mutations, and substitutions in the RPtag protein sequences may aid in producing additional binding enhancements.
Modifying Specificity of RPtag(Large) for Non RPtag(Small) Target Proteins (PDGF-β)
PDGF-β was selected as a target protein for these studies because of its clinical importance to diseases such as cancer and macular degeneration. In addition, the physically accessible N-terminus of PDGF-β possesses some homology to RPtag(small) (
An initial Kd was measured between RPtag(large) and the PDGF-β N-terminal peptide (
Mutation studies were next performed to identify residues in RPtag(large) that could enhance binding to PDGF-β peptide. For these studies, residues within 5 Å of the putative RPtag(small) binding cleft were identified—H122, D126, K129, G130, F137, A253, L233, and M270. Other positions within that area are I108, V120, S121, H122, I123, A124, S125, D126, K129, G130, M133, F137, F237, E241, L244, I247, K248, G250, A253, A254, T255, I256, A257, Q258, Q259, M263, L266, M270, K273, Y274, L275, and K276. Numbering for RPtag(large) for these studies is relative to “NativeRPtag(large)_with_Tags_and_Cys” provided below. This protein includes a 19 aa leader sequence (MGSSCHHHHHHSQDPNSSS). Next, 31 mutants of RPtag(large) were generated at a subset of 32 amino acids (H122, D126, K129, G130, F137, A253, L233, and M270) proximal to non-conserved residues between RPtag(small) and PDGF-β when modeled in the crystal structure. The Kd of each mutant was then measured to identify mutations that improved RPtag(large) affinity for PDGF-β peptide, but not for the a control RPtag(small) peptide (Nd2Cd4) (
These studies identified two positions where such mutations (with enhanced binding for PDGF-β peptide, but decreased binding for Nd2Cd4) occurred, H122 and A253. Two substitutions at these positions that provided the best affinity for PDGF-β peptide were H122L and A253R. This double mutant RPtag(large) exhibited a enhanced binding affinity for PDGF-β peptide (Kd=1.5 μM versus a Kd of 7.3 μM for binding to native RPtag(large)) and a reduced affinity for RPtag(small) (Nd2Cd4) (Kd=3.1 nM versus a Kd of 0.41 nM for binding to native RPtag(large)).
The modeled energy-minimized structure of the RPtag(large) H122L and A253R double mutant suggested a new hydrophobic interaction between PDGF-β peptide L5 and RPtag(large) peptide L122, and a new ion pair between PDGF-β peptide E15 and RPtag(large) peptide R253 (
This provides a plausible structural explanation for this tighter binding. Notably, the double mutant RPtag(large) bound purified PDGF-β dimer even tighter, with a Kd=75 nM. This further-enhanced binding is likely because of the proximity and corresponding high local concentration of two N-termini in the PDGF-β structure (
These studies show that RPtag(large) can be modified to select for binding to proteins other than RPtag(small) and its use as a monobody-type protein. One of skill in the art would understand that additional techniques may provide for further enhancements in both binding specificity and affinity of RPtag (large) for PDGF-β. For example, library based selection techniques such as phage and yeast display may be used to evolve RPtag (large) protein sequences with enhanced affinity for PDGF-β as well as other target proteins. Potential target proteins may include, without limitation, other PDGF isoforms, vascular endothelial growth factor (VEGF), VEGF receptor, erbB-2, HER2/neu, α-tubulin, amyloid-β (1-38, 1-40, 1-42 and other fragments), Estrogen receptors, TNF-α, Lupus anticoagulant antibodies, among others.
Materials and Methods
>NativeRPtag(large)_with_Tags_and_Cys (RPtagLarge sequence proper starts at MKEGKT . . . , all mutant numbering in here is done based off of this sequence including the tags and N-terminal Met.
MGSSCHHHHHHSQDPNSSSMKEGKTIGLVISTLNNPFFVTLKNGAEEKA
Regarding the studies shown in
The direct binding (left) and competition studies (right) presented in
As shown in
A standard binding equation was used to generate the plot in
Other methods are described in the text or in the figure legends.
While multiple embodiments are disclosed, still other embodiments of the present invention will become apparent to those skilled in the art from the following detailed description. As will be apparent, the invention is capable of modifications in various obvious aspects, all without departing from the spirit and scope of the present invention. For example, the experiments presented herein should not be construed to limit the mutations that can be introduced into RPtag(small) or RPtag(large) to alter their binding affinity or specificity. Accordingly, the detailed description is to be regarded as illustrative in nature and not restrictive.
Below is a Table, Table 4 showing some of the sequences used in these and other examples.
All references disclosed herein, whether patent or non-patent, are hereby incorporated by reference as if each was included at its citation, in its entirety. In case of conflict between reference and specification, the present specification, including definitions, will control.
Although the present disclosure has been described with a certain degree of particularity, it is understood the disclosure has been made by way of example, and changes in detail or structure may be made without departing from the spirit of the disclosure as defined in the appended claims.
This application claims benefit of priority pursuant to 35 U.S.C. § 119(e) of U.S. provisional patent application No. 62/468,323 filed on Mar. 7, 2017, U.S. provisional patent application No. 62/559,143, filed on Sep. 15, 2017, and U.S. provisional patent application No. 62/627,349, filed on Feb. 7, 2018, all of which are hereby incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/021385 | 3/7/2018 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/165328 | 9/13/2018 | WO | A |
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| 20060040327 | Amiss et al. | Feb 2006 | A1 |
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| 20140259212 | Plesch et al. | Sep 2014 | A1 |
| 20150010947 | Loh et al. | Jan 2015 | A1 |
| 20150185216 | Albert et al. | Jul 2015 | A1 |
| Number | Date | Country |
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| 2017066441 | Apr 2017 | WO |
| 2018076008 | Apr 2018 | WO |
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| Number | Date | Country | |
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| 20200102347 A1 | Apr 2020 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62627349 | Feb 2018 | US | |
| 62559143 | Sep 2017 | US | |
| 62468323 | Mar 2017 | US |
