HK1058365A - Process for folding chemically synthesized polypeptides - Google Patents

Description

Method for folding chemically synthesized polypeptides

Technical Field

The present invention relates to a method for folding chemically synthesized polypeptides. In addition, the present invention relates to a method for producing a biologically active protein.

Background

Since the successful chemical synthesis of fully active HIV-proteases, an enzyme consisting of 99 residues prepared by a highly optimized Solid Phase Peptide Synthesis (SPPS) method based on the standard Boc/Bzl method, the demand for synthetic proteins has been increasing in recent decades.

The 1994 synthesis of crystalline ubiquitin, a small molecule protein consisting of 76 residues, further indicated that high purity proteins can be synthesized by the SPPS method based on the Fomc/t-Bu scheme, which is operationally simpler and chemically less complex than the Boc/Bzl method.

Until 2000, there was a great deal of experimental evidence that single domain proteins containing 60 to 100 amino acid residues could be produced rapidly, reliably and economically by chemical synthesis with the aid of a peptide synthesizer in sufficient quantities for structural and functional studies.

Disulfide-bond containing proteins prepared by chemical synthesis, once folded, have the same properties as the native and genetically engineered forms of the protein. The disulfide bonds of proteins may form single or multiple intra-and/or inter-chain loop structures that impart a large amount of conformational restriction to the molecule and thus play a crucial role in stabilizing the biologically active conformation.

Structurally known single domain folded proteins can be prepared by regioselective pairing of cysteine residues. Cysteine protecting groups have been investigated in various combinations suitable for common protection schemes to allow fully selective deprotection and/or co-oxidation of cysteine residues in a step-wise and pair-wise manner.

However, the recent synthesis of an insulin-like peptide, human relaxin, indicates how complex the chemistry involved in the regioselective pairing of cysteine residues in proteins containing multiple cysteine residues is. The A chain precursor was synthesized by the SPPS method, the Fmoc/t-Bu method and the p-alkoxybenzyl alcohol-based resin, while the B chain precursor was synthesized by the Boc/Bzl method using PAM resin (4-carboxyamidomethylbenzyl ester linked to polystyrene-based resin). Two of the four cysteine residues of the A chain precursor are protected as S-Trt (S-triphenylmethyl) derivatives, while the other two cysteine residues are protected as S-Acm (S-acetamidomethyl) and S-Meb (S-p-methylbenzyl), respectively. The two cysteines in the B chain precursor are protected by S-Acm and S-Meb protecting groups. First, the intramolecular S-S bond of the A chain is formed by iodine oxidation in AcOH. Then, two intermolecular disulfide bonds connecting the a and B chains are formed by the following two steps: in the first step, the free thiol group obtained by HF deprotection of the S-Meb protecting group in the A chain precursor is reacted with the activated Cys (Npys) (S-3-nitro-2-pyridylsulfinyl) residue in the B chain (with the aim of forming an intermolecular heterologous disulfide bond), and in the second step the S-Acm group is removed by co-oxidation with iodine to obtain the remaining S-S bond.

The SPPS method offers the possibility of chemically synthesizing a variety of polypeptides containing cysteine residues protected with the same protecting group. Once the protecting groups are removed using a variety of oxidizing reagents, disulfide bonds can be formed directly. The treatment with iodine, N-iodosuccinimide and cyanogen iodide under such conditions minimizes the change in Tyr, Met and Trp which are sensitive to oxidation and prevents the sulfydryl group of cysteine from being oxidized to the corresponding sulfonic acid by carefully controlling the solvent, pH and reaction time, and results in efficient folding of polypeptides and/or proteins containing cysteine protected by S-Trt or S-Acm.

Thallium (III) trifluoroacetate can sometimes replace the above-mentioned oxidizing agents to give higher yields of disulfide bonds. The main drawbacks of this reagent are its toxicity, the difficulty of removing thallium from the target polypeptide and the need to protect Met and Trp residues from oxidation.

Oxidation reagents containing a mixture of sulfoxide/silyl compounds with trifluoroacetic acid have been successfully used to directly oxidize polypeptide precursors containing S-Acm, S-But, S-Met and S-Mob (S-p-methoxybenzyl) cysteine residues to form disulfide bonds. However, the use of this mixture is severely restricted by the need to protect the indole ring of Trp with a formyl group in order to avoid chlorination under oxidative conditions.

Methods for oxidative folding of synthetic linear polythiol precursors (reduced forms of polypeptides) are common and most frequently used. By this simplest method, the appropriate disulfide bonds can be formed naturally in the presence of air or some other mild oxidizing agent. Furthermore, the folding and cysteine pairing is accomplished in the presence of both Reduced (RSH) and oxidized (R-S-S-R) low molecular weight sulfhydryl compounds.

For synthetic polypeptides and small molecule proteins consisting of single domains, the thermodynamic driving force required for folding due to the combined effects of H-bonding, ion pairing and hydrophobicity is apparently sufficient to naturally produce the natural isomer during random denaturing oxidation reactions.

By conducting oxidative folding studies on small molecules containing multiple cysteines, such as enzymes, inhibitors, toxins or hormones, useful information is obtained about specific structural motifs, such as cysteine-stabilized β -turns, cysteine-stabilized poly (Pro) -II helix folds and cysteine-stabilized β - β -structure folds, where stabilization of the specific structural motif is the main driving force for the formation of the correct disulfide bond, even in smaller peptide molecules. If care is taken to select buffers, temperatures and additives that stabilize the secondary structural motifs, a completely correct folding of partially folded or irregular (misfolded) proteins is possible even in vitro.

In order to minimize the intramolecular cysteine mismatching that leads to the production of misfolded non-natural isomers and to avoid as much as possible the random formation of intermolecular disulfide bonds that promote aggregation and precipitation, various folding schemes have been devised that are suitable for the species of polythiol polypeptides.

Thus, it is common to air-oxidize the precursor in the form of the polymercapto at high dilution (1mg/ml or less than 1mg/ml) under neutral or slightly alkaline conditions. It usually takes a long time to continue the reaction and also produces a harmless by-product, namely water. However, it is difficult to control air oxidation because trace metal ions have a large influence on the air oxidation rate. More importantly, basic and hydrophobic precursor molecules tend to aggregate and precipitate out of solution at or near their basic or neutral isoelectric points during folding. Moreover, by-products generated by oxidation of Met accumulate during folding. Although the chemical operation steps required for folding the polythiol precursor are minimized, the formation yield of disulfide bonds promoted by the molecular oxygen of air is very low in many cases, and disulfide bonds cannot be formed at all sometimes.

DMSO and potassium ferricyanide have also been used as oxidizing agents. However, potassium ferricyanide must be used in the dark and, if Met and Trp are contained in the polypeptide chain, oxidative by-products accumulate during folding. The use of DMSO generally gives better results because it is effective in performing oxidative folding under acidic conditions without producing deleterious products in the reaction. The method is particularly suitable for folding of basic and hydrophobic polypeptide precursors due to their high solubility characteristics in acidic buffers. However, it is often reported that removal of DMSO from the final product presents difficulties and low selectivity for disulfide bond formation. Moreover, even with careful control of experimental conditions, the formation of random disulfide bonds, which lead to the production of misfolded isomers and oligomerization reactions, is always unavoidable.

The correct pairing and folding of cysteine in the polythiol precursor of small molecule proteins can be achieved in higher yields by using redox buffers such as oxidized (GSSG) and reduced (GSH) glutathione and cystine/cysteine (Cys/Cys).

Thus, during oxidative folding of GSSG/GSH or Cys/Cys induced ribonuclease A (R.R. Hantgan et al, Biochemistry (Biochemistry)13, 613, 1974), the 49 amino acid core domain of hirudin (B.Chatrenet and J.Y. Chang, J.Biol.chem. 267, 3038, 1992) and bovine trypsin inhibitor (BPTI) (T.E. Creighton, Methods enzymology (Methods Enzymol. 131, 83, 1986), free thiol and disulfide groups are formed and are constantly being remodeled throughout the folding process. Since the thiol/disulfide exchange reaction by the thiolate intermediate can facilitate the shuffling of the non-native disulfide bonds into native disulfide bonds, the overall rate and yield is generally higher than that of oxidative folding in air. For the case of oxidative folding in air, high dilution of the polymercapto precursor is necessary to avoid aggregation and oligomer and polymer formation and to maximize the yield of the target protein.

Hirudin^1-49In the first stage of in vitro folding, the unfolded reduced form (polymercapto) folds sequentially and irreversibly, forming equilibrium isomers containing one and two disulfide bonds and also equilibrium isomers containing three disulfide bonds (random isomers) (j.y. chang, journal of biochemistry (biochem. j).300, 643, 1994). Almost all 75 possible protein species, including the natural species, have been identified: 15 isomers with one S-S bond, 45 isomers with two S-S bonds and 15 isomers with three S-S bonds. In the second stage of folding, the random species are modified by shuffling the non-native disulfide bonds to obtain the native protein species. Disulfide bond formation is accelerated primarily by the use of oxidized glutathione or cystine, whereas disulfide bond shuffling requires a sulfhydryl catalyst such as reduced glutathione or cysteine or mercaptoethanol.

The effectiveness of the thiol reagents to promote shuffling is apparently related to their redox potential and each catalyst has an optimal concentration. During the accumulation of the irregular hirudin, cystine/cysteine was approximately 10 times more potent than GSSG/GSH. This difference has been explained by the relative redox potential between the GSSG/GSH (-0.24V) and Cys-Cys/Cys (-0.22V) systems. Hirudin is prepared by selecting a set of optimal conditions (temperature, buffer, salt and redox mixture)^1-49The folding process of (a) is accelerated to the extent that it can be completed in 15 minutes.

In general, the native conformation of a synthetic protein containing several disulfide bonds should form naturally under conditions best suited for folding of the polymercapto form. However, in many cases, even under optimal conditions, oxidative folding is mediated by the redox buffer described above, producing a large amount of by-products and mismatched forms. This is particularly the case for proteins that are prone to form in their native conformation only at specific membrane surfaces or with the aid of specific chaperones (s.sakakibara Biopolymers, Peptide Science 51, 279, 1999).

Moreover, although widely used, the oxidative folding reactions of the polythiol precursors facilitated by the air or GSSG/GSH and cystine/cysteine redox pairs have been most trially tried, as demonstrated by folding experiments for synthetic chemokines and chemokine analogs. In fact, although the native chemokines and their various analogs fold readily, the resulting folded structures are stabilized by two or three disulfide bonds, and several analogs do not fold fully under the same conditions under which the corresponding native molecules produce partially folded forms. These observations strongly suggest that changes in the primary structure of the polymercapto precursor may adversely affect the local correct folding (β -turns, polyproline helix motifs, etc.) in the polypeptide chain to be folded. Thus, the folding tendencies of many thiol precursors are primarily due to the intrinsic properties of the polypeptide chain and not to the function of the specific oxidative system acting on the molecule.

Enhancement of selective pairing of disulfide bonds by addition of alcohols, acetonitrile and DMSO to buffers of low ionic strength has also been reported. The protocol involves adjusting the electrostatic factors in the medium to favor the juxtaposition of oppositely charged amino acids that are adjacent to selected cysteine residues, thereby enhancing the formation of specific disulfide bonds.

Enzymes such as Peptidyl Disulfide Isomerase (PDI) and prolyl isomerase (PPI) have also been used as additives to catalyze and regulate disulfide conversion. If PDI is added to the refolding buffer, the time required to fold hirudin in vitro can be shortened to 10 hours to 30 seconds. In this case, the difference between the in vitro folding efficiency and the in vivo folding efficiency is not large.

When the cysteine residue is protected by an acid labile group such as Trt, the precursor of the polythiol polypeptide is directly obtained by acidolysis of the polypeptide-resin. Alternatively, preferably, the polypeptide in which all cysteines are protected by acid-resistant groups such as acetamidomethyl groups (Acm) is first isolated as an S-cysteine derivative by acid-splitting the polypeptide-resin, followed by treatment with Hg (AcO) in acetic acid₂Treatment was performed to remove the Acm groups, followed by removal of Hg ions by gel filtration in the presence of a large excess of mercaptoethanol.

However, in both cases, several side reactions have been reported for cysteine and tryptophan residues. The indole ring in tryptophan can be derivatized with mercaptoethanol and cysteine can produce a variety of side reactions, most importantly oxidation and alkylation reactions with t-butyl cations during the process of releasing the polypeptide chain from the resin by acid hydrolysis.

Therefore, due to the deficiencies of the prior art methods, there is a need for more efficient and simpler methods for folding chemically synthesized polypeptides and for preparing biologically active proteins by chemical synthesis.

Disclosure of Invention

It is therefore an object of the present invention to provide a simple, efficient and rapid method for folding polypeptides and/or proteins, wherein especially the production of isoforms containing mismatched disulfide bonds is minimized, and the use of expensive disulfide-shuffling reagents such as glutathione or enzymes can be omitted, and which is reproducible, stable and scalable. These and other objects will be apparent to those of ordinary skill in the art.

The present invention achieves this object by providing a method for folding chemically synthesized polypeptides, which comprises treating a polypeptide containing two or more derivatized cysteine residues with a reducing agent in a folding buffer having a predetermined pH and temperature.

Also provided is a method for producing a biologically active protein, the method comprising

(a) Chemically synthesizing a polypeptide comprising two or more derivatized cysteine residues;

(b) treating the polypeptide with a reducing agent in a folding buffer at a predetermined pH and temperature; and

(c) purifying the obtained folded protein.

Preferably, the derivatized cysteine residue corresponds to an S-butyl-thio-cysteine (S-t-Bu) residue. Thus, according to the present invention, it has unexpectedly been found that S-t-Bu-derived cysteines can be deprotected, i.e., the S-t-Bu moiety removed, and upon incubation in an appropriate folding buffer of appropriate temperature and pH, form disulfide bonds with other cysteines.

According to the invention, the reducing agent is preferably free cysteine. Excess cysteine may be added to the buffer (as shown in examples 1-5) or cysteine may be derived from the polypeptide (as shown in example 6).

In a preferred embodiment of the method of the invention, the folding buffer comprises one or more chaotropic salts, thereby bringing the peptide into equilibrium in which natural folding can occur. This can be achieved, for example, by subjecting the polypeptide and/or protein to conditions of complete denaturation, for example by using a high concentration of chaotropic salts, and then diluting the chaotropic salts to a lower concentration suitable for folding. The chaotropic salt is preferably selected from guanidine hydrochloride and urea, preferably present in a concentration of 0.1-1M during folding.

Preferably, the temperature of the folding buffer is between 25 ℃ and 40 ℃, more preferably between 27 ℃ and 38 ℃, in order to attenuate the change in the degradation of the peptide when compared to the natural body temperature. Most preferably the temperature during folding is about 37 deg.c.

According to another preferred embodiment of the method of the invention, the folding buffer has a weakly alkaline pH. Preferably, the pH is between 7 and 9, more preferably between 7 and 8.5 in order to facilitate the folding process. From the above it is clear that the folding of proteins depends on a complex set of interactions. For example, cysteine does not react at acidic pH, and higher pH values increase the risk of degradation of the polypeptide.

After folding, the target protein can be purified by methods known in the art, including anion and cation exchange chromatography, hydrophobic interaction chromatography, reverse phase chromatography, affinity chromatography, hydrophilic interaction/cation exchange chromatography (HILIC/CEC), Displacement Chromatography (DC), and sample displacement chromatography (SDM). Most preferred are (reverse phase) high performance chromatography and displacement chromatography using elution methods.

In a preferred embodiment of the method for producing a biologically active protein, the method comprises the steps of:

(a) assembling the S-tert-butyl-thiocysteine polypeptide onto an insoluble polymeric support by a stepwise chain extension method;

(b) cleaving the S-tert-butyl-thiocysteine polypeptide chain from the support by acid hydrolysis;

(c) purifying the obtained S-tert-butyl-thiocysteine polypeptide;

(d) folding the purified S-tert-butyl-thiocysteine polypeptide by treating the polypeptide with a molar excess of cysteine in a folding buffer containing a chaotropic salt, preferably guanidine hydrochloride, at an alkaline pH and about 37 ℃; and

(e) the obtained folded protein was purified by reverse phase high performance liquid chromatography.

In an advantageous embodiment of the process of the invention, the polymeric support is a polyamide or polystyrene-based resin functionalized with acid-labile hydroxymethylphenoxyacetic acid linkers, since these supports can be used in fully automated peptide synthesizers and are generally capable of synthesizing long polypeptide chains.

As explained above, the present invention is based on the stepwise solid-phase assembly of S-tert-butyl-thiocysteine polypeptides and the following findings: a large amount of the native biologically active protein can be produced by folding the polypeptide in the presence of a reducing agent, preferably cysteine, at slightly alkaline pH and at about 37 ℃.

It has surprisingly been found that biologically active proteins can be produced using a high molar excess of cysteine during the removal of the S-tert-butyl protecting group to form disulfide bonds. This procedure is simpler and more efficient than the procedures described in the prior art for folding cysteine-containing polypeptides, which are obtained by chemical synthesis. The removal of the S-tert-butyl group is thus accomplished in the same step as the folding of the polypeptide.

Alternatively, the same folded material can also be obtained by using a combination of S-tert-butyl-thiocysteine and cysteine protected by appropriate acid-labile groups that protect selected sites on the polypeptide chain so that an appropriate disulfide bond can be formed without the need for additional reducing agents to the folding buffer. In this case, the acid labile groups are removed while the peptide is cleaved from the resin at acidic pH. Free cysteine is thus produced which in turn acts as an intramolecular "reducing agent" capable of removing the S-tert-butyl group and forming disulfide bonds (as shown in example 6).

The essence of the invention lies in that:

-rapid assembly of the S-thio-tert-butylated polypeptide chain onto a polymeric support;

-cysteine catalyzes the thiol-disulfide bond exchange reaction of said derivative under weakly basic conditions, resulting in a cysteinylated polypeptide which is an oxidized macromolecular form of a typical redox cystine/cysteine pair (protein-S-S-cysteine; polypeptide-S-S cysteine);

the concentration of the oxidized macromolecular form is kept low throughout the folding process in order to minimize intermolecular disulfide exchange reactions; and

aggregation of misfolded intermediates does not occur due to preferential and rapid formation of the form (native structure) with the correct cysteine pairing.

According to the method for folding a polypeptide of the invention, for example, in a first step, 10mg of the S-tert-butyl derivative are dissolved at room temperature in 1ml of a buffer having a pH of 8.0 and containing 6M guanidine hydrochloride, 10mM Tris and 0.1M Na, and the resulting solution is left at room temperature for about 20 minutes₂HPO₄. In a second step, the solution was first diluted 10-fold with water to reach ph7.2, (0.6M guanidine hydrochloride,1.0mM Tris，10mMNa₂HPO₄and a final concentration of the polypeptide derivative of 1mg/ml), followed by the addition of a strong molar excess of cysteine (about 100-fold higher than the concentration of the polypeptide or protein derivative) under stirring conditions. To fold the polypeptide, the temperature was gradually raised to 37 ℃ and held constant for about 24 hours.

The folding method of the present invention is capable of producing a highly homogeneous product and requires only minor modifications to be applicable to any polypeptide produced by solid phase chemical synthesis, such as cysteine thio-tert-butyl derivatives. In addition, the process of the present invention using the form of the polythiol precursor has many other advantages over the prior art processes, such as

The cysteine residues in the chain are not alkylated during the acidolysis cleavage of the polypeptide-resin;

no peroxidation of cysteine to sulfonic acid, nor oxidation leading to intermolecular disulfide bond formation;

avoiding the risk of derivatization of the indole ring of Trp by mercaptoethanol, which is necessary for removing the Hg ions, which are a contaminant by means of Hg (AcO)₂Generated when performing Acm deprotection. In fact, cysteine mercaptides in the folding mixtures of the invention do not alter Trp at all;

the oxidation-sensitive Met, Trp and Tyr residues are not altered during folding;

the production cost of the final folded product is generally lower than that using a polythiol polypeptide and a redox buffer.

It is clear to those skilled in the art that although target proteins are usually prepared in high yield using the method of the present invention, in some cases, for example for complex proteins with multiple disulfide bonds, intermediate forms of a specific kind (misfolded species) that are not completely converted into the native structure remain in solution in an equilibrium state. Misfolded species can be conveniently separated from correctly folded species using RP-HPLC and refolded under the conditions of the present invention to increase the overall yield of the process.

According to the present invention, the term polypeptide refers to polymers of amino acids linked together by amide bonds. The term protein refers to a class of polypeptides having a three-dimensional structure, such as proteins present in cells and biological fluids of living organisms. Proteins, for example, can be composed of a single folded polypeptide chain or can be complex structures composed of multiple folded polypeptide chains.

The following examples and figures are provided to illustrate and not to limit the invention, which is limited in scope by the claims.

Drawings

FIG. 1 shows the HPLC profile before removal of the S-tert-butyl and folding of hu-I-309 (example 4).

FIG. 2 shows the results of mass measurement of the product of FIG. 1.

FIG. 3 shows an HPLC plot of deprotected folded hu-I-309 (example 4) indicating a shorter retention time.

FIG. 4 shows the results of mass measurement of the product of FIG. 3.

FIG. 5 shows the results of mass measurement of the product shown in FIG. 3 after treatment with NEM. In comparison with FIG. 4, no change in mass was observed, indicating the absence of free-SH groups.

FIG. 6 is a graph comparing the biological activity of recombinant I-309 with the biological activity of synthetic fold I-309 of the present invention. Biologically active by¹²⁵I-labeled chemokine binding to human lymphocytes.

FIG. 7 shows the analytical HPLC profile after protein folding in example 5.

FIG. 8 shows a preparative HPLC profile of the folded protein of example 5.

FIG. 9 shows the results of mass measurement of the purified product of example 5, showing that it has an expected molecular weight.

FIG. 10 shows an HPLC plot of the polypeptide of example 6 prior to S-tert-butyl removal and folding.

Fig. 11 shows the results of mass measurement of the polypeptide shown in fig. 10 (M ═ H).

FIG. 12 shows the HPLC profile of the protein of example 6 after folding, indicating a shorter retention time.

Fig. 13 shows the results of mass measurement of the protein shown in fig. 12 (M ═ H), indicating that it has the expected molecular weight.

Examples

Example 1Cys ^{10，11，34，50} Synthesis of (S-t-Bu) -hu-TARC (Thymus gland and activation-regulated chemokine) and folding

The chemokine derivative with 71 amino acid residues was assembled onto a 433A peptide synthesizer (Perkin Elmer/ABI) using Fmoc/t-Bu chemistry and polystyrene based resin functionalized with an acid labile hydroxymethylphenoxyacetic acid linker (Wang resin), to which Fmoc-Ser (t-Bu) was attached by esterification catalyzed by DMAP (4-dimethylaminopyridine). The degree of substitution was 0.57 mmole/g. The synthesis was carried out on a 0.27mmole scale by using five-fold excess of Fmoc-amino acid and DCI (N, N' -diisopropylcarbodiimide)/HOBt (1-hydroxybenzotriazole) activating reagent in DMF. The coupling reaction time was approximately 60 minutes with monitoring of Fmoc deprotection by spectrophotometry.

The four cysteine thiol groups were protected with S-tert-butyl groups and the maximum protection scheme was used for all other side chains: ser (t-Bu), Thr (t-Bu), Tyr (t-Bu), Asp (O-t-Bu), Glu (O-t-Bu), Lys (Boc), Trp (Boc), Asn (Trt), Gln (Trt) and Arg (Pmc). After each coupling, a capping reaction was performed with acetic anhydride and DIEA in DMF.

The resulting polypeptide-resin was treated with a freshly prepared TFA/water/TIS (triisopropylsilane)/phenol (78: 5: 12: 5, v/v/v/w, 10ml/g resin) mixture at room temperature for 2.5-3.0 hours. The cleaved polypeptide derivative was precipitated by filtering the cleavage mixture directly into cooled methyl-tert-butyl ether (MTBE), and the precipitate was separated by centrifugation, washed twice with diethyl ether and dried in air.

The crude product was then dissolved in diluted acetic acid, lyophilized, redissolved in 50% acetic acid and then loaded onto a Sephadex G-50 column (70X 25cm) with 50% acetic acid as the mobile phase. The collected fractions were analyzed by MALDI-TOF mass spectrometry and those containing the desired polypeptide derivative (MW8, 436.9Da) were combined, diluted with water and lyophilized.

The combined fractions were redissolved in 50% acetic acid and loaded into 250X 10mm semi-preparative Vydac C₄Further purification was performed on a chromatographic column. The sample was eluted over 60 minutes using a linear gradient of 20-80% B in 0.1% TFA acetonitrile and a in 0.1% TFA water at a flow rate of 3 ml/min. Detection was performed at 280nm, and only the fractions containing the target polypeptide were pooled and lyophilized prior to folding.

The folding reaction of the chemokine derivatives purified by RP-HPLC was carried out according to the following procedure: 10mg of the product are first dissolved in 1mg of 6MGnHCl, 0.1M Na at pH8.0 and room temperature₂HPO₄And 10mM Tris. After 20 minutes, the solution was diluted to a final concentration of 0.6M GnHCl, 10mM Na by adding 10ml of water₂HPO₄1mM Tris, pH7.2 and peptide concentration 1mg/m 1. The folding reaction was initiated by adding cysteine at a concentration of about 20mM (about 100-fold molar excess relative to the peptide concentration) and the temperature was gradually raised to 37 ℃.

Aliquots of 25 microliters of solution were performed by using a Waters2690 separation moduleSamples were subjected to RP-HPLC analysis using a Waters996 photodiode array detector to monitor folding reactions in air at a constant temperature of 37 deg.C, with the aliquots being acid quenched with acetic acid, and the RP-HPLC analysis using a Vydac C₄The column was analyzed and elution was performed for 40 minutes with a 20-60% acetonitrile gradient in 0.1% TFA/water and a flow rate of 1.0 ml/min. 1 microliter of each HPLC peak eluate (corresponding to the folding intermediate of the thiol-disulfide exchange reaction) was collected and then mixed with 1 microliter of a saturated solution of sinapic acid dissolved in 1: 2 acetonitrile/1% TFA in water, dried in vacuo and analyzed by MALDI-TOF mass spectrometry using a Voyager-DE spectrometer (Perselective Biosystem, Framingham, Mass.) equipped with a nitrogen laser. 78% of the folded polypeptide was formed after 24 hours. The presence of free thiol groups (+ 125Da for each SH) was detected by further examining the peaks whose molecular weight corresponds to that of the folded product by reaction with N-ethylmaleimide (NEM).

The biological activity of hu-TARC obtained by the method of the present invention was measured according to the Imai method (T.Imai et al, J.Biol.chem., 271, 21514, 1996).

Human T cell lines, Hut78, Hut102 and Jurkat, as well as fresh monocytes, neutrophils and lymphocytes were assessed for migration through polycarbonate filter paper in response to TARC. Neither TARC, produced by chemical synthesis nor recombinant TARC, elicits chemotactic responses in monocytes or neutrophils. In the T cell lines Hut78 and Hut102, migration induced by synthetic and recombinant TARCs exhibited a typical bell-shaped curve with the strongest induction at 100 ng/ml.

Example 2Cys ^10，34，50 (S-t-Bu) -hu-TARC and Cys ^11，34，50 Synthesis of (S-t-Bu) -hu-TARC And folding

Cys is performed^10，34，50(S-t-Bu) hu-TARC and Cys^11，34，50Synthesis of (S-t-Bu) -hu-TARC derivative,Conditions and Cys used for purification and folding^{10，11，34，50}The same conditions were used for (S-t-Bu) hu-TARC (example 1), the only difference being that Cys was individually treated with Cys¹⁰And Cys¹¹Trt protection is performed and the protecting group is removed at the same time as cleaving the polypeptide precursor from the resin. The final yields of folded chemokines were 80% and 79%, respectively.

Example 3CVs ^34，50 Synthesis and folding of (S-Bu) -hu-TARC

Except that Trt is used for Cys¹⁰And Cys¹¹In addition to protection, Cys is performed^34，50The synthesis, purification and folding of the (S-Bu) -hu-TARC derivatives were carried out under the same conditions as those used for the derivatives of examples 1 and 2, and the Trt was removed during the final resin cleavage with TFA. The yield of folded product was about 75%.

Example 4Cys ^{10，11，26，34，50，68} Synthesis and folding of (S-t-Bu) -hu-I-309

hu-I-309 containing 6 cysteines protected with (S-t-Bu) was synthesized on a 0.12mmole scale using Fmoc-Lys (Boc) Wang resin (degree of substitution of 0.61 mmol/g) under the same conditions as in example 1. The resulting polypeptide-resin was treated as described in example 1, and then purified by loading G50 into 250X 10mmVydac C₁₈For further purification on the column (as shown in FIGS. 1 and 2).

By dissolving 65mg of the product in 60ml of 0.6M GuHCl, 10mM NaHPO, pH8.0₄And folding the RP-HPLC purified chemokine derivative in 1mM Tris and cysteine in a 100-fold molar excess over the peptide. The polypeptide solution was maintained at 37 ℃ for 4 days. After acidification with TFA, 250X 10mmVydac C was used₁₈The column separates the folded material by RP-HPLC (as shown in fig. 3 and 4). Complete cysteine pairing was verified by mass spectrometry after reaction with N-ethylmaleimide (NEM). No increase in molecular weight was observed, indicating the absence of free thiolsA radical group (as shown in figure 5). The yield of folded final chemokine was close to 25%. The synthetic folded hu-I-309 has biological activity equivalent to that of the recombinant protein (FIG. 6).

Analytical chromatography was performed according to the following conditions: a chromatographic column: c₁₈250X 4.6mm (Vydac #238TP54) mobile phase: a is 100% H₂O0.1％TFA

B＝100％CH₃CN 0.1% TFA gradient: the% composition of B is shown on the chromatogram. A detector: 214nm

Example 5Synthesis and folding of C-terminal fragments of Plasmodium vivax

Plasmodium vivax circumsporozoite protein (PvCS)303-372 containing 4 (S-t-Bu) -protected cysteines was synthesized and purified under the same conditions as in example 1.

Folding was performed by adding 27mg of the peptide to 2.7ml of 6M GuHCl in 0.1M Tris buffer (pH 8.5). The solution was stirred for 10 minutes. 13.5ml of 1mM EDTA, 0.2M NaCl, pH buffered at 8.8 with 0.2M Tris buffer was then added. Finally, 10.8ml of 35mM cysteine/1 mM EDTA, 0.2M NaCl buffered at 8.8 pH with 0.2M Tris buffer was added. The reaction mixture was warmed to 37 ℃. The folding reaction was then performed by reverse phase HPLC (3-6 hours) (fig. 7A) and terminated by cooling at 4 ℃ for 5 minutes, then a final concentration of 1% TFA (3ml of 10% TFA) was achieved by adding 4 ℃ 10% TFA. The product was then purified by reverse phase HPLC (fig. 8) and the quality of the final product was determined (fig. 9). The yield of oxidized final product is 70-80%.

Analytical chromatography was performed using the following conditions: a chromatographic column: c₄250X 4.6mm (Vydac #214TP54) mobile phase: a is 100% H₂O0.1％TFA

B＝100％CH₃CN 0.1% TFA gradient: the% composition of B is shown on the chromatogram. A detector: 214nm

Example 6Plasmodium falciparumLarge-Scale Synthesis of (Plasmodium falciparum) C-terminal fragments and folding

Large-scale synthesis and purification of Plasmodium falciparum circumsporozoite protein (PfCS282-383) containing only 2 of the 4 (S-t-Bu) -protected cysteines were carried out under the same conditions as in example 1, except for the following conditions.

By dissolving 1.1g of partially purified peptide in 1.0L of 0.1MCH at pH8.0₃COONH₄Without the need to add free cysteine to the folding buffer. The reaction mixture was maintained at 32 ℃ for 18 hours. The product was then purified by reverse phase HPLC (fig. 12 and 13). The yield of oxidized final product is close to 37%.

Analytical chromatography was performed using the following conditions: a chromatographic column: c₁₈250X 4.6mm (Vydac #238TP54) mobile phase: a is 100% H₂O0.1％TFA

B＝100％CH₃CN 0.1% TFA gradient: the% composition of B is shown on the chromatogram. A detector: 214nm

Claims (27)

1. A method for folding a chemically synthesized polypeptide comprising treating a polypeptide and/or protein having two or more derivatized cysteine residues with a reducing agent in a folding buffer having a predetermined pH and temperature.

2. The method of claim 1, wherein the derivatized cysteine residue corresponds to an S-butyl-thio-cysteine residue.

3. The method of claim 1 or 2, wherein the reducing agent is cysteine.

4. The method of claim 1, 2 or 3, wherein the folding buffer comprises one or more chaotropic salts.

5. The method of claim 4, wherein the chaotropic salt is selected from the group consisting of guanidine hydrochloride and urea.

6. The method of claim 4 or 5, wherein the chaotropic salt in the folding buffer is present at a concentration of 0.1-1M.

7. The method of any one of claims 1-6, wherein the folding buffer has an alkaline pH.

8. The method of claim 7, wherein the pH is between 7 and 9.

9. The method of claim 7 or 8, wherein the pH is between 7 and 8.5.

10. The method of any one of claims 1-9, wherein the folding buffer is at a temperature between 25 ℃ and 40 ℃.

11. The method of claim 10, wherein the temperature is between 27 ℃ and 38 ℃.

12. The method of claim 10 or 11, wherein the temperature is about 37 ℃.

13. A process for preparing a biologically active protein comprising

(a) Chemically synthesizing a polypeptide comprising two or more derivatized cysteine residues;

(b) treating the polypeptide with a reducing agent in a folding buffer at a predetermined pH and temperature; and

(c) purifying the obtained folded polypeptide and/or protein.

14. The method of claim 13, wherein the derivatized cysteine residue corresponds to an S-butyl-thio-cysteine residue.

15. The method of claim 13 or 14, wherein the reducing agent is cysteine.

16. The method of claim 13, 14 or 15, wherein the folding buffer comprises one or more chaotropic salts.

17. The method of claim 16, wherein the chaotropic salt is selected from the group consisting of guanidine hydrochloride and urea.

18. The method of claim 15 or 16, wherein the chaotropic salt in the folding buffer is present at a concentration of 0.1-1M.

19. The method of any one of claims 13-18, wherein the folding buffer has an alkaline pH.

20. The method of claim 19, wherein the pH is between 7 and 9.

21. The method of claim 20, wherein the pH is between 7 and 8.5.

22. The method of any one of claims 13-21, wherein the folding buffer is at a temperature between 25 ℃ and 40 ℃.

23. The method of claim 22, wherein the temperature is between 27 ℃ and 38 ℃.

24. The method of claim 22 or 23, wherein the temperature is about 37 ℃.

25. A method according to any of claims 13-24, comprising the steps of:

(a) assembling the S-tert-butyl-thiocysteine polypeptide onto an insoluble polymeric support by a stepwise chain extension method;

(b) cleaving the S-tert-butyl-thiocysteine polypeptide chain from the support by acid hydrolysis;

(c) purifying the obtained S-tert-butyl-thiocysteine polypeptide;

(d) treating the polypeptide derivative with a molar excess of cysteine in a folding buffer comprising a chaotropic salt and having an alkaline pH and about 37 ℃ to fold the purified S-tert-butyl-thiocysteine polypeptide; and

(e) the obtained folded protein was purified by reverse phase high performance liquid chromatography.

26. The method of claim 25, wherein the chaotropic salt is guanidine hydrochloride.

27. The method of claim 25 or 26, wherein the polymeric support is a polyamide or polystyrene based resin functionalized with an acid labile hydroxymethylphenoxyacetic acid linker.