Patent Application
20250066831
The contents of the electronic sequence listing (156P663sequence.xml; Size: 12,521 bytes; and Date of Creation: Jun. 20, 2024) is herein incorporated by reference in its entirety.
The present invention related to lipidated protein complexes and, more specifically, to an approach for producing farnesylated proteins from genetically engineered bacteria.
The development of new protein-based biopharmaceuticals and materials is a vibrant area of research at the interface of chemistry, biology, and materials science and engineering. Efforts in this space have traditionally focused on engineering the amino acid sequence and structure of proteins to achieve a specific function for applications including biomaterial, sensors, and biocatalysts, among others. Instead of changing the sequence of proteins, cells leverage an alternative strategy, post-translational modification (PTMs), to modulate a protein's structure and function with exquisite spatiotemporal control. Despite the significant interest in the biology of PTMs, this strategy remains underutilized for diversifying physicochemical properties, engineering capabilities, and/or the biological behavior of proteins. This lacuna exists because the synthesis of proteins with compositionally defined PTM patterns remains challenging, and although more than 300 PTMs have been identified, the energetic interplay and (bio) material consequences of only a handful of these modifications have been systematically studied.
Lipidation is a common PTM in eukaryotes where the nature of the attached lipid (e.g., fatty acids, sterols, isoprenoids, etc.) dictates biological outcomes by controlling protein structure, function, and localization. For instance, isoprenylation of Ras signaling proteins—the modification of cysteine residues with either 15 carbon (farnesyl) or a 20 carbon (geranylgeranyl) isoprenoid lipid—is critical for their localization to correct membrane-bound organelles, as well as regulating their function. While the biology of isoprenylation and the interactions between prenylated proteins and lipid bilayer are of broad research interest, application of this PTM to control the assembly and function of protein-based materials remains underexplored. This is due in large part to the lack of a facile, scalable, and inexpensive synthetic method to lipidate proteins. Many lipidated proteins (LP) cannot be produced by genetic code expansion methods due to the stringent preference of ribosomes for amino acid-derived motifs. Alternatively, their multi-step convergent semi-synthesis is laborious and technically challenging. The inability to rapidly generate LPs has hindered the empirical elucidation of sequence-structure-function rules and computational parametrization of LPs' design space. Accordingly, there is a need in the art for an operationally simple, high-yield biosynthetic route to produce prenylated proteins.
The present invention is an operationally simple, high-yield biosynthetic route to produce prenylated proteins by genetically engineering E. coli to co-express the desired protein and the minimum enzymatic machinery required for prenylation. In eukaryotes, isoprenylation is carried out by specialized transferases that bind to a lipid donor (isoprenyl pyrophosphate) and modify a recognized peptide substrate fused to the C-terminus of target proteins. The present invention focuses on farnesylation because E. coli can biosynthesize farnesyl pyrophosphate (FPP) from the sequential condensation reactions of dimethylallyl pyrophosphate with two molecules of 3-isopentenyl pyrophosphate. This endogenous metabolic pathway provides access to farnesyl lipid donors without the need for genetic/metabolic engineering. The next requirement for the construction of the minimal recombinant expression system for protein farnesylation is the identification of appropriate prenyl transferase enzyme(s) that can be heterologously expressed in an active form in bacteria. Three major classes of prenyl transferases are known: farnesyltransferase (FTase) and geranylgeranyl transferase (GGTase) types I and II. The present invention included an investigation into the potential utility of FTase and GGTase-I due to their broad substrate scopes: proteins fused to the “CaaX” (SEQ ID NO: 1) motif at the C-terminus. In this motif, the cysteine, which is the site of PTM, is followed by two hydrophobic amino acids; and the final amino acid (X) determines preferential selectivity toward FTase (X=Ser) or GGTase-I (X=Leu).
To reveal how farnesylation modulates the assembly and properties of proteins, elastin-like polypeptides (ELPs) were as a model system. ELPs are artificial peptide polymers that are derived from the consensus sequence of tropoelastin (GZGVP), in which the guest reside (Z) can be any amino acids other than proline. ELP was chosen as model system for two reasons: 1) ELPs have a well-characterized lower critical solubility transition (LCST) behavior in which they undergo a soluble-to-insoluble temperature above a critical transition temperature (Tt). In addition to solution parameters (e.g., concentration, ionic strength, pH, etc.), this Tt depends on the molecular syntax of the ELP (e.g., the hydrophobicity of the guest residue, the length of the polypeptide) and the physicochemistry of PTM motif. The phase-separation behavior of ELP can be conveniently monitored using various scattering techniques and therefore enable us to parse the effect of farnesylation on the thermo-response of lipid-protein conjugates. 2) ELPs can be expressed at high yields in E. coli and can be purified at scale using non-chromatography techniques that leverage their reversible temperature-triggered phase behavior. Additionally, the stimuli-responsive characteristics of ELPs enable the fine-tuning of the emergent assembly of farnesylated protein with temperature.
An embodiment of the present invention thus includes a recombinant organism for forming a lipidated protein, where the recombinant organism is a host organism that has been modified to include a first gene expressing an alpha subunit and a beta subunit of a transferase that will attach a lipid from a lipid donor to a protein. The host organism is further modified to include a second gene that expresses the protein, wherein the protein includes a substrate of the transferase. The transferase may be selected from the group consisting of farnesyltransferase and geranylgeranyl transferase. The substrate of the transferase may comprise a first amino acid that is cysteine, a second amino acid that is hydrophobic, a third amino acid that is hydrophobic, and a fourth amino acid that has selectivity to farnesyltransferase or geranylgeranyl transferase. The host may be capable of endogenously producing the lipid donor. The lipid donor may comprise farnesyl pyrophosphate. The substrate of the transferase may be selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3. The alpha subunit and the beta subunit may be translationally coupled, such as by a stop codon of the beta subunit overlapped with a start codon of the alpha subunit. The host organism may comprise a bacterium such as E. coli.
In another embodiment, the present invention may be a method of producing a lipidated protein. In one step, the method involves modifying a host organism to include a first gene expressing an alpha subunit and a beta subunit of a transferase that will attach a lipid from a lipid donor to a protein. In another step, the method involves modifying the host organism to include a second gene expressing the protein, wherein the protein includes a substrate of the transferase. In a further step, the method involves providing a lipid donor. In an additional step, the method involves culturing the host organism to express the first gene and the second gene in the present of the lipid donor to form the lipidated protein. The transferase may be selected from the group consisting of farnesyltransferase and geranylgeranyl transferase. The substrate of the transferase may comprise a first amino acid that is cysteine, a second amino acid that is hydrophobic, a third amino acid that is hydrophobic, and a fourth amino acid that has selectivity to farnesyltransferase or geranylgeranyl transferase. The host may be capable of endogenously producing the lipid donor. The lipid donor may comprise farnesyl pyrophosphate. The substrate of the transferase may be selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3. The alpha subunit and the beta subunit may be translationally coupled, such as by a stop codon of the beta subunit overlapped with a start codon of the alpha subunit. The host organism may comprise a bacterium such as E. coli.
The present invention will be more fully understood and appreciated by reading the following Detailed Description in conjunction with the accompanying drawings, in which:
Referring to the figures, wherein like numerals refer to like parts throughout, there is seen in
The present invention employed an iterative process to reconstitute and optimize the protein-farnesylation platforms. As a proof-of-concept example, endogenously produced FPP was used as a lipid donor to minimize the number of exogenously expressed proteins to three: a model protein (i.e., ELP) was fused to the CaaX sequence (SEQ ID NO: 1), and the alpha and beta subunits of FTase or GGTase-I. Both FTase and GGTase-I are heterodimeric proteins with an identical a subunit but divergent β subunits. FPP is the canonical lipid donor for FTase, but it is also accepted by GGTase-I. Two prenyl transferase subunits were selected from R. norvegicus as they have been previously expressed in E. coli. Genes encoding a and β subunits as well as a model ELP fused to short peptide sequences, CVLS (SEQ ID NO: 2) and CVLL (SEQ ID NO: 3) were cloned into a set of orthogonal plasmids with compatible origins of replication (pBR322 and p15A) and selection markers (Amp′ and Cm′) using Gibson assembly and recursive directional ligation and their identity were verified by DNA sequencing (
To identify conditions for high-yield heterologous production of the transferase, the pETDuet-1 vector was initially used to co-express the prenyltrasferase subunits in BL21(DE3) strains. This bicistronic vector uses two independent T7/lac promotors to independently produce alpha and beta subunits. Despite the high expression efficiency of this vector, almost all the expressed transferase was found in insoluble inclusion bodies, as seen in
The laborious and costly synthesis of FPP hinders the in vitro production of farnesylated proteins at a scale sufficient for various biomedical and material engineering applications. Therefore, it was investigated whether co-expression of protein substrate and the transferase can be used to recombinantly produce desired farnesylated proteins in a one-pot expression-lipidation system as seen in
Having established that one-pot recombinant expression of farnesyl-modified proteins is possible, the conditions to maximize yields of farnesylated proteins was systematically characterized. The efficiency of farnesylation in E. coli is influenced by concentration of the available intracellular pool of FPP, the binding constant of FPP to each prenyl transferase, and the rate of lipid transfer to the protein of interest, which is influenced by accessibility of the CaaX motif (SEQ ID NO: 1), and the identity of “X”-residue. Since a priori estimation of these variables is difficult, it was determined the amount of farnesylated proteins using a 2×2 factorial design with prenyl transferase (FTase and GGTase-I) and the identity of X-residue (Ser and Leu).
Large scale protein expression and purification of both V40-Fr and (V/A)80-Fr (and their nonlipidated controls) were conducted according to protocols described in the supporting information. Briefly, plasmids encoding the α- and β-subunits of GGTase-I and substrate protein fused to CVLL (SEQ ID NO: 3) were co-transformed to BL21 (DE3) E. coli strains. The bacteria were cultivated at 37° C. to OD600˜0.8. At this point, the temperature was reduced to 28° C. and the expression culture was supplemented with ZnSO4 (0.5 mM, metal cofactor required for GGTase-I), and the co-expression of GGTase-I and polypeptide fused to the enzyme recognition sequence was induced by IPTG (1 mM). After 18 h, cells were harvested by centrifugation. The ELPs were isolated by adapting a method recently developed by Thompson and coworkers to purify post-translationally lipidated ELPs. This approach uses a combination of organic (non) solvents to lyse the cells and to selectively isolate the ELP and hybrid biopolymers (as shown in this paper) from the complex mixture of cellular proteins.
After purification, a combination of HPLC, MALDI-TOF MS, and NMR was used to verify the identity and purity of constructs, and to establish that only one farnesyl group is added to the protein at the cysteine residue located in the C-terminal CaaX motif (SEQ ID NO: 1), as seen in
For both ELPs, the molecular ion peak corresponding to lipidated protein was shifted by m/z=+205.2 Da, consistent with the addition of a farnesyl motif (and removal of a hydrogen atom from the thiol). In each case, the doubly charged ion [M+2H]2+ was detected, as seen in
Because V40-Fr lacked a trypsin digestion site near the farnesylation site, 1H NMR spectroscopy was used to confirm its farnesylation. The spectra of V40 and V40-Fr in D2O is shown in
Finally, FT-IR was used to see if lipidation perturbs the structure of ELPs by comparing the amide absorption bands of unmodified and farnesylated V40 (
Biopolymers with programmable thermo-response (such as ELPs) are attractive materials for biomedical applications because temperature can be increased locally as a therapeutic modality while causing minimal damage to healthy tissues. While the effect of amino acid mutations on the liquid-liquid phase separation of proteins is under intense investigation, our understanding of the effect of lipidation on the phase-boundaries remains incomplete. Given the importance of thermo-response in biomedical applications, we used complementary techniques of turbidimetry and DSC to quantify the effect of farnesylation on the temperature-triggered phase separation of elastin-based proteins.
To determine whether the phase separation of farnesyl-modified proteins is reversible, the turbidity of the solution was monitored as the temperature was reduced from 65 to 15° C. with the cooling rate of 1° C./min (blue curves). As shown in
The effect of farnesylation on the concentration dependence of Tt was also evaluated for both constructs. Quantifying this behavior is critical for the development of models that can reliably predict the behavior of farnesylated constructs in biomedical applications in which the concentration of protein changes as a function of time, (e.g., thermally triggered drug depots, intravenous administrations, or hyperthermia-based targeting and treatment of cancer). Turbidimetry was used to measure the transition temperature for unmodified and farnesylated proteins at various concentrations (3, 6, 10, 12.5 μM in PBS), as seen in
To investigate how farnesylation modulates the thermodynamics of liquid-liquid phase separation, we used DSC to measure the total heat of phase separation.
As discussed earlier, the broad peaks in the NMR (
The visual inspection of ACFs for farnesyl-modified proteins also shows that the exponential decay profile is not monotonic, which likely is due to equilibrium between assemblies of different size. The bimodal size-intensity distributions at different angles (13°, 90°, and 173°) shown in
To determine the effect of temperature on the properties of these assemblies, DLS was conducted from 15-65° C. Initially, temperature steps were set at 5° C., but smaller temperature steps (0.5-1° C.) were applied within Tt (turbidimetry)±5° C. The ACF at each temperature was analyzed using the method of cumulants to calculate the average hydrodynamic diameter of proteins at each temperature (
Cryo-TEM was used to image the assemblies of farnesylated proteins at 20° C. (below their transition temperature). Intriguingly, noticeable differences was observed in the nanostructure of V40-Fr and (V/A)80-Fr, even though DLS yielded similar Zavg for both constructs. As shown in
Referring to
By converging synthetic biology with materials science, the present invention provides recombinant platforms to produce farnesylated proteins with programmable assembly and temperature-dependent characteristics. Although in vitro and in lysate prenylation of proteins has been previously demonstrated, the laborious and expensive synthesis of prenyl donors limits the scalability of these methods. Consequently, they are typically used to produce small quantities (a few μg) of naturally occurring farnesylated proteins for biochemical characterization or chemoenzymatic labeling of proteins with bio-orthogonal handles. The method 10 of the present invention enables scalable production of farnesylated proteins outside of the biological context (i.e., with artificial proteins and/or noncanonical lipidation sites) for biomaterial and biomedical applications. The present invention also captures the differences between closely related prenyl transferases for efficient modification of proteins in these recombinant platforms. Seminal biochemical studies had previously established that GGTase-I can accept both FPP and GGPP as the lipid donor, and the identity of the X-residue in CaaX (SEQ ID NO: 1) box alters the substrate-preference of transferase. Nonetheless, the present invention reveals that the concentration and availability of prenyl donors inside a microbial factory are different from the conditions often used to characterize the biochemistry of enzymes using large excess of lipid or peptide substrate. The present invention also demonstrates that the identity of the X-residue determines whether farnesylated proteins can self-assemble into recombinant nanoparticles (if X is hydrophobic amino acids such as valine) or not (when X is hydrophilic amino acids such as serine),
By combining biophysical and soft-matter characterization techniques, it has been shown that farnesylation alters the thermo-response, phase separation, and assembly of ELPs used as model protein system. These effects are consistent with the physicochemistry of the farnesyl group, as this hydrophobic lipid increases the unfavorable interactions of ELP with water and modulates the energetics of the phase separation via the supramolecular oligomerization of farnesyl-modified proteins. Dynamic light scattering and cryo-TEM show that the farnesylated assemblies likely exist in a dynamic equilibrium between unimers and assembled structures. The chemical structure of farnesyl, which contains three unsaturated bonds, is likely responsible for the reduced hydrophobicity and increased dynamics of micellar assemblies. The differences observed between the properties of farnesylated proteins vs. proteins modified with saturated fatty acid or sterols strongly suggest that the physicochemical properties of the lipid can be used as a design principle to control the biophysical and material properties of this class of hybrid materials.
Recent biochemical studies have greatly expanded the size of potential prenylated proteome beyond the classic CaaX motifs (SEQ ID NO: 1) to other motifs such as C(x)3X and Cxx. Nonetheless, the correlation between the sequence of these lipidation sites and the assembly or localization of prenylated proteins remains less clear. The present invention sets the stage for future studies to reveal the interplay of prenylation sites or the effect of post-prenylation processing steps on the emergent properties of farnesylated proteins. Moreover, the strategy of the present invention can be applied to the biosynthesis of geranylgeranylated proteins by metabolically engineering E. coli to increase the intracellular GGPP. These studies are underway in our laboratories and will be reported in due course.
Cloning. Genes encoding α and β subunits of FTase and GGTase-I (from Rattus norvegicus) as well as their canonical substrate peptides, CVLS (SEQ ID NO: 2) and CVLL (SEQ ID NO: 3) were constructed using standard molecular biology techniques. All cloning steps were conducted using E. coli EB5α strain. The identity of each gene was confirmed by Sanger sequencing. After verifications, plasmids were co-transformed to E. coli BL21(DE3) for protein expression. See Table 1 below for additional details.
A bacterial colony was used to inoculate a flask containing 50 mL of sterile 2×YT medium supplemented with kanamycin (45 mg/mL). After overnight growth in a shaking incubator (37° C., 200 rpm, 16 h), 4 mL of this culture was used to inoculate 1 L of 2×YT media. The bacteria were grown in an orbital shaker incubator at 37° C. at 200 rpm. The expression was induced by adding isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM when the optical density reached 1.5. After 18 hours, cells were harvested by centrifugation (3745×g, 4° C. for 30 min). The bacterial pellet was resuspended in phosphate buffer saline (PBS, pH 7.4, 10 mL/L of expression culture). The cells were stored at −80° C. until purification.
A 50 mL of sterile 2×YT medium, supplemented with ampicillin (100 mg/mL) and chloramphenicol (25 mg/mL), was inoculated with a bacterial colony and incubated in an orbital shaker (37° C., 200 rpm) to OD600 of 1.5. 4 mL of this media was used to inoculate each one liter of 2×YT medium. The bacteria were grown in an orbital shaker incubator at 37° C. at 200 rpm. After reaching to OD600 of 0.8, the temperature was reduced to 28° C. and the expression was induced by adding IPTG and ZnSO4 to the final concentrations of 1 mM and 0.5 mM, respectively. After continuing the expression at 28° C. for 18 h, the cells were harvested by centrifugation (3745×g, 4° C., 30 min). The bacterial pellet was resuspended in PBS (pH 7.4, 5 mL/L of expression culture).
Proteins were purified by adapting a recently reported method for rapid purification of ELPs for isolation of post-translationally lipidated proteins. Briefly, the cell pellets were incubated with isopropanol, which lyses the cells and precipitates most endogenous proteins and nucleic acids. After separation of insoluble debris, the expressed ELPs are precipitated by adding a non-solvent, acetonitrile to the final volume of 70% (v/v). The protein pellet was resuspended in 50% (v/v) ethanol in water and purified by preparative RP-HPLC to ensure >95% purify for characterization studies.
Preparative and analytical RP-HPLC were performed on a Shimadzu instrument equipped with a photodiode array detector on C18 columns (Phenomenex Jupiter® 5 μm C18 300 Å, 250×4.6 mm and 250×10 mm). A mobile phase consisting of gradient of acetonitrile and water (supplemented with 0.1% TFA) was used to elute the proteins, as seen in Table 2 below:
The thermal response of proteins was analyzed using a Cary 100 UV-Vis Spectro-photometer (Agilent, Santa Clara, CA) equipped with a Peltier temperature controller. The absorbance of protein solutions at 350 nm were continuously monitored between 15-65° C. while heating/cooling the solution at the rate of 1° C./min.
The molecular weight of proteins was determined using MALDI-TOF-MS, conducted on a Bruker Microflex® LRF instrument. To determine the location of farnesyl group, the proteins were digested with trypsin, and the peptide fragments were analyzed using MALDI-TOF-MS.
MADLS was performed using Zetasizer Ultra (Malvern Instruments, UK) at the scattering angles of 13°, 90°, and 173°. Protein solutions (6 μM in PBS) were filtered into a DLS cuvette and analyzed at 15-65° C. Measurements were performed in triplicate after incubating the samples for 3 min at each temperature. Scattering autocorrelation functions were analyzed with Zetasizer software using the cumulant method to derive average hydrodynamic radius (Zavg) and polydispersity index. The intensity-size distributions are calculated using CONTIN method.
Protein solutions were applied to a freshly plasma cleaned grid (Pelco easiGlow, negative polarity, 45 s, 30 mA) and plunged frozen in liquid ethane. Grids were stored under liquid nitrogen until they were imaged on a Tecnai BioTwin 120 kV transmission electron microscope, operated at LN2 temperature. Samples were imaged under low-dose conditions using a Gatan 626 or a Gatan 910 holders, cooled to LN2 temperature. Images were collected on a Gatan SC1000A CCD-camera. TEM images were analyzed using ImageJ.
NanoDSC (TA instruments, New Castle) was used to quantify the enthalpy of phase-separation by measuring the excess heat capacity of the protein solution (against PBS reference) while heating the sample 10 to 65° C. at a rate of 1° C./min.
The FT-IR absorption spectra were collected using Thermo Scientific Nicolet iS5 FT-IR Spectrometer with iD7 attenuated total reflectance accessory by sandwiching the lyophilized proteins directly over the crystal. The spectral resolution was set to 4 cm-1 and each spectrum were obtained with 128 scans.
1H NMR spectra were recorded with Bruker Avance III HD 400 MHz. The samples were prepared by dissolving lyophilized proteins into deuterium oxide or dimethyl sulfoxide-d6 at the concentration of 1.67 mg/mL. The proton NMR spectra were collected at 25° C.
The pACYCDuet-I vector was purchased from EMD Millipore (Billerica, MA). The chemically competent Eb5α and BL21(DE3) cells, restriction enzymes, ligase, and corresponding buffers, as well as DNA extraction and purification kits, were purchased from New England Biolabs (Ipswich, MA). Isopropyl β-D-1-thiogalactopyranoside (IPTG) was purchased from A. G. Scientific (San Diego, CA). Apomyoglobin, adrenocorticotropic hormone (ACTH), sinapinic acid, alpha-cyano-4-hydroxycinnamic acid, zinc sulfate, and trifluoroacetic acid (TFA) were purchased from Sigma-Aldrich (St. Louis, MO). High-performance liquid chromatography-(HPLC) grade acetonitrile, SnakeSkin™ dialysis tubing with 3.5K nominal molecular weight cut off (MWCO), mass spectroscopy grade Pierce™ trypsin protease, tryptone, yeast extract, sodium chloride, ampicillin, kanamycin, chloramphenicol, phosphate buffer saline (PBS), DMSO, isopropanol, acetonitrile, and ethanol were purchased from Thermo Fisher Scientific (Rockford, IL). Mini-PROTEAN® TGX Stain-Free™ Precast Gels, Precision Plus Protein™ All Blue Pre-stained Protein Standard, and Precision Plus Protein™ Unstained Protein Standards were purchased from Bio-Rad Laboratories, Inc. (Hercules, CA). Deionized water was obtained from a Milli-Q® system (Millipore SAS, France). Simply Blue™ SafeStain was purchased from Novex (Van Allen Way Carlsbad, CA). All chemicals were used as received without further purification.
The sense and antisense oligonucleotides encoding for the canonical peptide substrate of FTase (CVLS) (SEQ ID NO: 2) and GGTase-I (CVLS) (SEQ ID NO: 2) were purchased from IDT DNA. The 5′-ends of single-stranded DNAs were phosphorylated, after which the complementary oligos were thermally annealed. The double-stranded DNA was then cloned into a modified pET24a(+) vector, referred to as pJMD5, which had been double digested with BseRI and BamHI. Recursive directional ligation by plasmid reconstruction was used to fuse the peptide substrates to the C-termini of V40 and (V/A)80 genes.1 After sequence verification, the ELP-CVLS (SEQ ID NO: 2) or ELP-CVLL (SEQ ID NO: 3) were cloned into pACYCDuet-I using NdeI and XhoI restriction enzymes. Genes encoding for a and b subunits of FTase-I and GGTase-I were ordered as gene fragments from IDT DNA and were cloned into pETDuet-1 using Gibson assembly. The beta subunit was cloned into MCS1 between NcoI and EcoRI sites, and the alpha subunit was cloned into MCS2 between Nde and XhoI. The construction of vectors used for translational coupling of a and b subunits has been reported previously.
EF
‘QEEF’ at the end is translated from the RBS site that is proceeding the alpha
subunit start codon in the translationally-coupled system (
The proteins were purified by optimizing a recently reported extraction method of ELP. The method was optimized to apply for lipidated proteins as well as to reduce the isolation time to 40 min. The cells were pelleted by centrifuging at 21850×g at 4° C. for 5 min. After decanting the supernatant, the cell pellet was resuspended in isopropanol (4 mL/g of the wet pellet). After thorough mixing by vortexing and bath sonication for 5 min, the cells were further mixed with isopropanol by rotating for 5 min at 25° C. The protein was then separated in the supernatant by centrifuging at 15000×g for 5 min at 25° C. The protein was then precipitated by adding acetonitrile to the final composition of 70% (v/v). The solution was then centrifuged at 15000×g for 5 min at 25° C. After discarding the supernatant, the protein pellet was resuspended in 50% (v/v) ethanol in water. The suspension was centrifuged at 15000×g, 25° C. for 5 min to remove any insoluble impurities. The protein was then analyzed by Reverse-phase HPLC (RP-HPLC). After organic extraction, the solution contained both unmodified and farnesylated ELPs. The lipidated product can be separated from the unmodified product by leveraging its lower transition temperature. The farnesylated proteins were then purified by preparative HPLC to ensure purity (>95%) for characterization studies. RP-HPLC was performed with a Shimadzu HPLC system (Phenomenex Jupiter® 5 μm C18 300 Å, LC Column 250×10 mm, solvent A: H2O+0.1% TFA, solvent B: acetonitrile+0.1% TFA). The percentage of the organic solvent in the mobile phase was increased from 0 to 90% over the course of 23 minutes. After HPLC purification, the organic solvent was removed by dialysis against water using SnakeSkin™ Dialysis Tubing (3500 MWCO, Thermo Scientific) overnight, followed by lyophilization. Lyophilized proteins were stored at −20° C.
Analytical RP-HPLC was performed on a Shimadzu instrument using a Phenomenex Jupiter® 5 μm C18 300 Å, 250×4.6 mm LC Column with a mobile phase consisting of a gradient of acetonitrile in water containing 0.1% trifluoroacetic acid (Table 2) to analyze the proteins. The proteins were analyzed using a photodiode array detector at wavelengths between 190 and 230 nm.
MALDI-TOF-MS was conducted on Bruker Microflex® LRF with a microScout ion source. A saturated solution of sinapinic acid in 50% acetonitrile was used as the matrix. The Samples were prepared by mixing 3 μL of 25 μM protein solutions with 7 μL of the matrix followed by serial dilution. These solutions were plated onto a sample plate and dried at room temperature. Apomyoglobin (Mw=16,952.27 Da) was used as standard.
To identify the location of the farnesyl group, (V/A)80-Fr were digested with trypsin, and the peptide fragments were analyzed using MALDI-TOF-MS. To set up the reaction, 9 μL of protein (100 μM) was added to 10 μL of 100 mM ammonium bicarbonate buffer (pH=7.8) in an Eppendorf tube. The reaction was initiated by adding 1 μL trypsin (reconstituted as 5 μg/μL in 50 mM acetic acid) at 37° C. After 3 h, the peptide fragments were analyzed by MALDI-TOF-MS. α-cyano-4-hydroxycinnamic acid was used as the matrix for the analysis of the trypsin-digested peptide fragments. The instrument was calibrated using adrenocorticotropic hormone (Mw=2,464.1989 Da).
Temperature-triggered phase separation studies of the proteins were performed with an Agilent UV-Vis Spectrophotometer (Cary100) equipped with a Peltier temperature controller by measuring the absorbance of the solution at 350 nm. Four concentrations (3, 6, 10, and 12.5 μM in PBS) of proteins were analyzed by heating the solution at the rate of 1° C./min from 15 to 65° C. For reversibility studies, the protein solutions were then cooled to 15° C. at the same rate. The Transition temperature (Tt) was defined as the inflection point, i.e., the maximum of the first derivative, in the absorbance during the heating cycle. These data were fitted to the following model Tt=−m×ln [protein]+Tc to derive critical transition temperature (Tc) and the concentration dependence of Tt (m), summarized in Table 3 below.
[a]° C./ln (μM/μM). 95% confidence intervals are calculated from the linear regression analysis using Graphpad prism.
The present application claims priority to U.S. Provisional Application No. 63/292,234, filed on Dec. 21, 2021.
This invention was made with government support under Grant Nos. R01GM132606 and R35GM142899 awarded by the National Institutes of Health (NIH) and Grant No. 2105193 awarded by the National Science Foundation, Division of Materials Research. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US22/82119 | 12/21/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63292234 | Dec 2021 | US |
