Method for space packing chemically synthesized polypeptides

FIELD: chemistry of polypeptides.

SUBSTANCE: invention relates to a method for space packing (folding) of chemically synthesized polypeptides. Method comprises treatment of polypeptide and/or protein comprising two or more S-butyl-thiocysteine residues with cysteine in buffer providing the folding process at average pH value from 7 to 9 and at temperature in the range from 25°C to 40°C.

EFFECT: improved method for space packing.

19 cl, 13 dwg, 6 ex

 

The present invention relates to a method of spatial packing chemically synthesized polypeptides. In addition, the present invention relates to a method for production of biologically active proteins.

During recent decades there has been increasing demand for synthetic proteins obtained in the course of a successful chemical synthesis of fully active HIV protease enzyme, consisting of 99 residues, which is obtained as a result of implementation of optimized methods of solid-phase peptide synthesis (CFPS) based on application of the standard approach with the Boc/Bzl protection.

Synthesis in 1994 crystalline ubicacin, a small protein consisting of 76 residues, further demonstrated by the fact that high-purity proteins can be synthesized by methods TFPS based procedures Fmoc/t-Bu, a method that is easier to implement and chemically is a less complex process than the procedure Boc/Bzl.

By 2000 had accumulated enough experimental evidence that proteins with the same domain containing from 60 to 100 amino acid residues, can be obtained quickly, reliably and cost-effectively by chemical synthesis using a peptide synthesizer in quantities sufficient for the study of structural properties and functional activity of the.

Proteins containing disulfide bridges, produced by chemical synthesis, by folding have the same properties as natural forms and forms received genetic engineering methods. Disulfide bridges of proteins form a single or multiple intra - and/or megamachine cyclic structures, which give the conformation of the molecules is a significant limitation that has a decisive impact on the stabilization of the biologically active conformation.

Folded proteins of known structure with a single domain can be obtained by regioselective coupling to cysteine residues. In combination with commonly used protection procedures have been developed various combinations of protective agents for cysteine groups that are compatible with the existing methods, which allow for speed and pairwise removal of protective groups and/or cookilaria cysteine residues with full compliance selectivity.

However, a recent synthesis of insulin-like peptide - human relaxin - showed how difficult and therefore demanding the chemistry underlying the regioselective coupling cysteine residue in proteins containing multiple cystine residues. The synthesis of the precursor chain And was conducted in the context of the methodology TFPS with the use of what Finance Fmoc/t-Bu-approach and resin-based p-alkoxybenzyl alcohol, and the predecessor of the b-chain was obtained using resin PAM (of ester 4-carboxymethylamino attached to the resin based on polystyrene) followed by Boc/Bzl-procedure. Of the four cysteine residues of the precursor And-circuit protection two of them were conducted in the form of derivatives of S-Trt (S-triphenylmethyl), while others were selected security options in the form of S-Acm (S-atsetamidometil) and S-Meb (S-p-methylbenzyl), respectively. The protection of the two cysteines in precursor b-chain was performed using the protective groups of S-Acm and S-Meb. Intramolecular S-S bridge in the a-chain was received first in the oxidation of iodine in the Asón. Then during two stages received two intermolecular disulfide bridge connecting the chains a and b: in the first stage, the free thiol precursor chains And obtained by HF-releasing the protective group S-Meb, was subjected to reaction with activated Cys(Npys) (S-3-nitro-2-pyridylsulfonyl) balance-chain (directed through the formation of intermolecular getarticleid), and the second stage received the remaining S-S-bridge by sookielee remove groups of S-Acm with iodine.

Procedures TTPS allow to obtain by chemical synthesis of a variety of polypeptides containing cysteine residues protected the same block is their protective groups. After removal of the protective groups using a variety of known oxidants are formed directly disulfide bonds. Polypeptides and/or proteins containing cysteine protected using S-Trt or S-Acm can be effectively minimized by treatment with iodine, N-yogakriya and cyanoguanidine in strictly controlled conditions, including choice of solvent, pH and reaction time, which allows to minimize the modification of the oxidation-sensitive residues Tyr, Met, and Trp and to avoid excessive oxidation of the cysteine-thiol to the corresponding sulfonic acids.

Triptorelin thallium(III) may be substituted for the above oxidants with achievement sometimes even the best outputs disulfide bonds. The main limitations for the specified reagent is its toxicity, difficulty of removal of thallium from the desired polypeptide and the need to protect the remnants of Met and Trp in the oxidation process.

For direct oxidation with the formation of disulfide bonds of polypeptide precursors containing S-Acm, S-But, S-Meb and S-Mob (S-p-methoxybenzyl) derivatives of cysteine residues, have been used successfully oxidants containing a mixture of sulfoxide/silyl compounds and triperoxonane acid. However, the main limitation for the application of this mixture is the need to protect the indole what about the ring of Trp with formyl order to prevent chlorination in oxidative conditions.

Methods of oxidative folding synthesized linear Polterovich predecessors (the recovered polypeptide forms) are the most known and used most often. In the case of the simple method corresponding disulfide bond can be spontaneously formed in the presence of air or some other mild oxidizing agents. In addition, folding and pairing of cysteine residues is achieved in the presence of reduced (RSH), and oxidized (R-S-S-R) forms low molecular weight sulfhydryl compounds.

In synthetic polypeptides and small proteins consisting of a single domain, thermodynamic driving force that carries out the folding, which is a merger of H-bonding, ion pairing and hydrophobic effects, obviously is quite sufficient for the spontaneous formation of native isomers in random conditions of oxidative renaturation.

The study of oxidative folding of small proteins containing multiple cysteine, such as inhibitors of enzymes, toxins or hormones, was obtained useful information about certain structural motifs, such as the cysteine-stabilized β-turn, cysteine-stabilized poly(Pro)II helical folding and cysteine-stabilizer the EN α -β-structural folding, stabilization, which is the main driving force for the correct formation of disulfide bonds even in a relatively small peptide molecules. And if you pay enough attention to the selection of buffers, temperature, and additives, capable of stabilizing the secondary structural motifs,in vitrocan be achieved even completely correct folding and partially folded or improperly folded proteins.

We developed many procedures to minimize Polterovich polypeptides to minimize cases of incorrect intramolecular pairing cysteine, which leads to the production of non-native, correctly folded isomers, as well as to avoid, to the extent possible, education random intermolecular disulfide bonds, which accelerates the aggregation and precipitation.

Thus, the air oxidation is usually carried out at high dilution palaiologoi forms predecessor (1 mg/ml or below) under conditions of neutral or slightly alkaline environment. This usually requires a long period of time and leads to the formation during the reaction harmless by-product in the form of water. However, the oxidation air is difficult to control due to the fact that trace amounts of metal ions strongly affect the speed of these reactions is. More importantly, basic and hydrophobic molecules predecessors are prone to aggregation and precipitation from solution in conditions close to their main or neutral isoelectric points during the process of folding. Furthermore, in the folding process produces by-products associated with the oxidation of methionine. Although the number of chemical operations required to collapse Polterovich predecessors, minimized the formation of disulfide bonds, which contributes to molecular oxygen, leading in many cases to low output, and sometimes the desired product is not formed.

As oxidizers were also used DMSO and potassium ferricyanide. However, the potassium ferricyanide you want to use in the dark, and if in the polypeptide chain contains methionine and tryptophan, in the course of the folding process are accumulated by-products of oxidation. The use of DMSO often gives the best results due to the fact that oxidative folding can be carried out in acidic conditions with high speed and without formation in the reaction of harmful products. The method is particularly suitable for minimizing basic and hydrophobic polypeptide precursors due to higher solubility of those types, to the which undergo oxidation in acidic buffers. Often, however, reported problems associated with the removal of DMSO from the finished product and with the decrease in the selectivity of the formation of disulfide bridges. In addition, violation of order disulfide bonds leads to incorrect isomers, it is impossible to avoid oligomerization even with careful control of experimental conditions.

High levels of correct mating cysteine and collapse Polterovich predecessors small proteins often are achieved with the use of redox buffers, such as oxidized (GSSG) and reduced (GSH) glutathione and cystine/cysteine (Cys/Cys).

Thus, during the oxidative folding of ribonuclease A (R.R. Hantgan et al., Biochemistry 13, 613, 1974), nuclear domain of hirudin, consisting of 49 amino acids (b Chatrenet and J.Y. Chang, J. Biol. Chem. 267, 3038, 1992), and inhibitor of bovine pancreatic trypsin (BPTI) (T.E. Creighton, Methods Enzymol. 131, 83, 1986), induced with GSSG/GSH or Cys/Cys, constantly producing and conversion of free sulfhydryl and disulfide groups in the whole process of collapse. General speed and outputs of the reactions in this case are usually higher than during the oxidative folding in the air, because the thiol/disulfide exchange taking place with the participation of intermediate derivatives of tialata, facilitate the AET shuffling ranatunga disulfide in its natural form. As in the case of oxidative folding in the air, you need high dilution politologe predecessor to eliminate aggregation, the formation of oligomers and polymers and to achieve maximum yields of the target protein.

At the first stage of collapse of hirudin1-49in vitrocollapse occurs consistently, and permanently, since resveratol restored form (politial), before the formation of an equilibrium mixture of isomers containing one and two disulfide bridge, and before the formation of an equilibrium mixture of products containing three disulfide bonds (unordered isomers) (J.Y. Chang, Biochem. J. 300, 643, 1994). Were identified almost all of the 75 possible types of protein, including native form: 15 isomers with one S-S bridge, 45 isomers with two S-S bridges and 15 isomers with three S-S bridges. In the second stage of the folding of disordered types of protein are restructuring by shuffling negativnyh disulfides with the formation of native protein forms. The formation of disulfide accelerated mainly oxidized glutathione or cystine, whereas for disulfide shuffling required thiol catalyst, for example restored glutathione, or cysteine, or mercaptoethanol.

It is obvious that the efficiency of thiol reagents in terms of ease of shuffling is related to their oxidation in stenopetalum potential, and each of the catalysts shows the optimum concentration. In the process of accumulation of disordered girginov cystine/cysteine is about 10 times stronger effect than GSSG/GSH. This difference can be explained by the relative value of the redox potential of the pair GSSG/GSH (-0,24) and a pair of Cys-Cys/Cys (-0,22). When choosing the optimal combination of conditions (temperature, buffer, salts, and redox mixture) the phasing out of hirudin1-49is accelerated to such an extent that it is completed within 15 minutes.

In General, the native conformation of a synthetic protein containing disulfide bonds must be formed spontaneously under conditions optimal for minimizing Polterovich forms. However, in many cases, even in the optimized conditions under oxidative folding when using the above redox buffers, produce large amounts of by-products and forms with the wrong mate. This is particularly true in the case of those proteins, which have a tendency to the formation of the native conformation only on the surface of specific membranes or using specific molecular chaperone (S. Sakakibara, Biopolymers, Peptide Science 51, 279, 1999).

In addition, despite the widespread use, most prozessoptimierung collapse Polterovich predecessors under the influence of air or redox pairs GSSG/GSH and cystine/cysteine is made by trial and error, that was clearly demonstrated in the experiments to minimise the synthetic chemokine and chemokine analogues. In fact, while native chemokines and many of their counterparts collapses easily, and their folded structure stabilized by two or three disulfide bridges, some analogs do not give a good collapse in the same conditions that were used in case of appropriate native molecules, forming a partially folded form. These observations give strong reasons to believe that changes in the primary structure Polterovich predecessors may have an adverse effect on the induction of proper education local folded forms (α-turns, polyproline spiral motifs and others) in a collapsible polypeptide chains. In this regard, the ability to minimize many of thiol precursors represents, above all, the internal property of the polypeptide chain, and not a function of specific oxidative system, acting on the molecule.

About the strengthening of education selected disulfide pairs adding alcohols, acetonitrile and DMSO to the buffers with low ionic strength is also reported in the literature. This strategy is based on strengthening the education of specific disulfide bonds by adjusting the electrostatic factors in the environment, FPIC is stuudy desired location adjacent oppositely charged amino acids, they border the selected cysteine residues.

Enzymes, such as peptidyltransferase (SOPS) and preliminaires (PI), were also used as additives for catalysis and modulation disulfide exchange. The time required for the folding of hirudinin vitrocan be reduced from 10 hours to 30 seconds, if the buffer is to minimize add SOPS. In this case, the efficiency of foldingin vitronot significantly different from the situation observedin vivo.

Politologie polypeptide precursors directly receive when algorithmics.com splitting complex polypeptide-resin in the case where cysteine residues protected colorability groups, for example, Trt. Alternative and preferably, polypeptides, in which all cysteine protected acid group, for example acetamidomethyl group (Acm), first isolated in the form of S-cysteine derivatives by algoritmicheskoe cleavage complex peptide-resin, after which the Acm group is removed by treatment with Hg(AcO)2in acetic acid, followed by removal of Hg ions by gel filtration in the presence of a large excess of mercaptoethanol.

It is reported, however, that in both cases, cysteine and tryptophan residues have some adverse reactions. Indole ring of tryptophan can be the ü modified-mercaptoethanol, and in the case of cysteine has a number of adverse reactions, the most important of which are the oxidation and alkylation cations tert-butyl during algoritmicheskoe remove the polypeptide from the resin.

Thus, due to the shortcomings of existing methods there is a need for more efficient and simpler ways to minimize chemically synthesized polypeptides, and biologically active proteins by chemical synthesis. On this basis, the aim of the present invention to provide an efficient, simple and fast way folding of polypeptides and/or proteins, in the exercise of which, including, minimizes the formation of isomers containing incorrect disulfide bridges do not need to use expensive reagents for the shuffling of disulfide bonds, such as glutathione or enzymes, and this method is reproducible, simple, and scalable. These and other objectives will become apparent to experts in this field.

This goal is achieved by the present invention through the development of a method of folding a chemically synthesized polypeptide, which includes the processing of the polypeptide that contains two or more derivatizing cysteine residue in the restoration of the Ghent in the buffer to collapse, having a given pH and temperature.

In addition, we propose a method of obtaining biologically active proteins, including

(a) chemical synthesis of the polypeptide that contains two or more derivatizing cysteine residues;

(b) processing the specified polypeptide regenerating agent in the buffer to minimize having a specified pH value and temperature; and

(C) clearance obtained folded proteins.

Preferably derivationally cysteine residue corresponds to residue S-butyltoluene (S-t-Bu). Thus, in accordance with the present invention, the authors found that S-t-Bu-derived cysteine can be subjected deprotection, i.e. they may lose S-t-Bu-grouping with the formation of disulfide bridges with other cysteine in the case of incubation in the appropriate buffer to collapse under suitable temperature and pH.

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

In a preferred embodiment of the present invention the buffer for minimizing includes one or more chaotropic salts with C is poured bring polypeptide in the equilibrium state, which promotes natural spatial packing. This goal can be achieved, for example, by placing the polypeptide and/or protein in conditions of full denaturation, e.g. due to high concentration chaotropic salts with subsequent dilution chaotropes salt to a low concentration for the implementation of collapse. Chaotrope salt is preferably selected from the group consisting of chloride guanidine and urea, and preferably they are present in a concentration of 0.1-1 M in the process of collapse.

Preferably the temperature of the buffer to collapse lies in the range from 25 to 40°S, more preferably from 27 to 38°With, in order to reduce changes in the degradation of the peptide, but to conform to the natural temperature of the body. Most preferably the temperature in the process of folding is approximately 37°C.

According to another preferred variant implementation of the present invention the buffer to collapse has a slightly alkaline pH. Preferably the pH value were made in the range from 7 to 9, more preferably from 7 to 8.5, in order to facilitate the process of collapse. As can be seen from the previous description, the folding of a protein depends on a complex set of interactions. For example, the reaction with cysteine is not at acidic pH, and bol is e high pH values increase the risk of degradation of the polypeptide.

After completion of the folding of target proteins can be purified well known in the art methods, including anyone - and cation-exchange chromatography, chromatography based on hydrophobic interaction chromatography with reversed phase, affinity chromatography, chromatography, based on hydrophilic interaction/casinoonline (HILIC/CEC), pressure chromatography (I) and displacement chromatography of the sample (WMO). Most preferably the use of high-performance chromatography (reversed-phase) with elution and displacement chromatography.

In a preferred embodiment of the invention for the production of biologically active proteins this method includes a stage

(a) Assembly S-tert-butyl-Tolstonogov polypeptide in an insoluble polymeric substrate through a step-by-step chain elongation;

(b) removal of the specified S-tert-butyl-tolstonog polypeptide chains with specified substrate through acidolysis;

(C) purification of the obtained S-tert-butyl-Tolstonogov polypeptide;

(d) folding the purified S-tert-butyl-Tolstonogov polypeptide by processing the specified polypeptide molar excess of cysteine in the buffer to collapse, including chaotrope salt, preferably the chloride guanidine, and having selon the e value of pH and a temperature of about 37° With; and

(e) purification of the obtained folded proteins by high-performance liquid chromatography with reversed phase.

In a successful embodiment of the method according to the present invention, the polymer substrate is a polyamide or a resin based on polystyrene, functionalized cyclotourism linker in the form of hydroxymethyltransferase acid, as these substrates can be successfully used for fully automated peptide synthesizers and basically allow the synthesis of long polypeptide chains.

As noted above, the present invention is based on a sequential solid-phase Assembly of S-tert-butyl-tolstonog polypeptide and detecting the fact that a significant number of native biologically active proteins can be obtained by conducting the collapse of these polypeptides in the presence of a reducing agent, preferably cysteine, when the pH value is slightly above neutral and at a temperature of about 37°C.

It was unexpectedly discovered that the formation of biologically active proteins is achieved using a high molar excess of cysteine in the procedure of removal of S-tert-butyl protective group, which is accompanied by the formation of disulfide bonds is astika. This procedure is more simple and effective than described on the level of equipment procedures for the folding of cysteine-containing polypeptides produced by chemical synthesis. Thus, the removal of S-tert-butyl performed on the same stage as the folding of the polypeptide.

Alternatively, the same folded material can be obtained by using the combination of S-tert-butyl-Tolstaya and cysteine, is protected by acid-labile groups in the selected provisions of the polypeptide chain in order to enhance the education of the corresponding disulfide bonds without the need for excessive adding a reducing agent to the buffer to collapse. In this case, the acid-labile group is deleted when the peptide otscheplaut from the resin under acidic pH. The formation of free cysteine, which, in turn, act as intramolecular "reducing agent", which ensures the removal of S-tert-butyl and the formation of disulfide (as shown in example 6).

The essence of the present invention is based on

- quick Assembly S-thio-tert-bottled polypeptide chains on the polymeric substrate;

is catalyzed by cysteine thiol-disulfide exchange derivatives in slightly alkaline conditions, which leads to obtaining cysteinemia the different polypeptides, representing macromolecular oxidized form (protein-S-S-cysteine; polypeptide-S-S-cysteine) classical redox couples cystine/cysteine;

- maintaining the concentration of oxidized macromolecular shape constant at a low level during the whole process of folding, so that is minimized intermolecular disulfide exchange; and

- the absence of aggregation incorrectly folded intermediate products with fast and preferred structures with the correct pairing of cysteine (native structures).

According to the method of folding of the polypeptides according to the present invention, for example, in the first stage 10 mg of S-tert-Putilkovo derived dissolved at room temperature in 1 ml of buffer with pH 8.0, containing 6 M of chloride guanidine, 10 mm Tris and 0.1 M Na2HPO4and the resulting solution was incubated at room temperature for about 20 minutes. In the second stage, the solution is first diluted 10 times with water to a pH of 7.2 (0.6 M chloride guanidine, 1.0 mm Tris, 10 mm Na2HPO4at final concentration derived polypeptide 1 mg/ml), and then add with stirring a large molar excess of cysteine (approximately 100 times higher concentrations derived polypeptide or protein). The temperature of the mixture gradually increased to 37°and under eribaum constant for about 24 hours, to have passed the folding of the polypeptide.

The method of folding according to the present invention results in vysokogomogennogo product and is applicable with minor modifications to any polypeptide obtained by solid-phase chemical synthesis in the form of thio-tert-butyl derivative of cysteine. Furthermore, the method according to the present invention has many other additional advantages in comparison with the methods available at that level of technology, which is used politologie form of a precursor, such as

- cysteine residues circuit does not alkiliruyutza in the process algoritmicheskoe cleavage complex polypeptide-resin;

- does not occur pereokislenie cysteine to sulfonic acid or oxidation, leading to the formation of intermolecular disulfide bridge;

- eliminates the risk of modification of the indole ring of tryptophan-mercaptoethanol, which is necessary for removal of pollutants Hg ions formed during the release of Acm using Hg(AcO)2. In fact, tiolet cysteine in mixture to collapse according to the present invention not modify tryptophan;

- sensitive to oxidation residues methionine, tryptophan and tyrosine are not modified in the process of folding;

- the cost of production of finished rolled products in the foundations of the om below in comparison with the cost methods, using politologie polypeptides and redox buffers.

Specialists in this field it is obvious that despite the fact that the target protein is basically possible to obtain according to the method of the present invention with high yield, in some cases, for example, in the case of complex proteins with multiple disulfide bonds in the solution in equilibrium will remain a certain proportion of intermediate forms, which have not undergone the transition to the native structure (incorrectly folded structures). Such incorrectly Packed spatial patterns can be easily separated from the properly Packed structures by HPLC-PF and again placed in terms of folding according to the present invention to improve the overall yield of the process.

According to the present invention, the term polypeptide refers to a polymer composed of amino acids linked together by an amide bonds. The term protein refers to a polypeptide structure in its three dimensional form, which occurs in cells and biological fluids of living organisms. Proteins can, for example, consist of a single folded polypeptide chains or can be a complex structure consisting of many folded polypeptide chains.

The following examples and drawings are given for illustrate the present invention and are not intended to limit the invention beyond those limitations, which imposes on him the claims.

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

Figure 2 shows the result of the determination of the mass of the product is shown in figure 1.

Figure 3 shows the HPLC profile freed from the protective groups collapsed hu-I-309 (example 4), demonstrating a shorter retention time.

Figure 4 shows the result of the determination of the mass of the product is shown in figure 3.

Figure 5 shows the result of the determination of the mass of the product is shown in figure 3, after processing NEM. In comparison with figure 4 is not observed mass change, which indicates the absence of free-SH groups.

6 is a graphical illustration of a comparison of the biological activity of recombinant human I-309 and synthetic I-309, minimized according to the present invention. Biological activity was assessed by binding of the chemokine labeled125I, with human lymphocytes.

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

On Fig shows the profile preparative HPLC folded protein described in example 5.

Figure 9 shows the result of the determination of the mass of the purified product from example 5, indicating the presence of the expected molecular weight.

Figure 10 shows the HPLC profile of the polypeptide of example 6 preduzimanjem S-tert-butyl and collapse.

Figure 11 shows the result of the determination of the mass of the polypeptide shown in figure 10 (M=N).

On Fig shows the HPLC profile of the protein from example 6 after folding, demonstrating a shorter retention time.

On Fig shows the result of the determination of the mass of the protein, shown in Fig (M=N), indicates the presence of the expected molecular weight.

Examples

Example 1

Synthesis and folding Cys10,11,34,50(S-t-Bu)-hu-TARC

(thymus and activation regulated chemokine)

Derived chemokine, consisting of 71 amino acid residue, subjecting the Assembly to a peptide synthesizer 433 And (Perkin Elmer/ABI) using technology Fmoc/t-Bu and resin based on polystyrene containing a functional group obtained by treatment cyclotourism linker based hydroxymethyltransferase acid (resin Wang; Wang resin)to which Fmoc-Ser(t-Bu) connected via the esterification reaction catalyzed by DMAP (4-dimethylaminopyridine). The degree of substitution is 0.57 mmol/g Synthesis is carried out on a scale increments 0.27 mmol, using a five-fold excess of Fmoc-amino acids and reagents, activated with a mixture of DCI (N,N'-diisopropylcarbodiimide)/HOBt (1-hydroxybenzotriazole), in DMF). Binding time is approximately 60 minutes to conduct spectrophotometric monitoring of deprotect Fmoc.

Four of cysteine thiol Conn who are using S-tert-Budilnik groups and use the scheme maximum protection 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 binding conducting coating acetic anhydride, DIEA in DMF.

The resulting complex polypeptide-resin is treated at room temperature with freshly prepared mixture of TFU/water/TIS (triisopropylsilane)/phenol (78:5:12:5, vol/vol/volume/weight, 10 ml/g resin) for 2.5 to 3.0 hours. Derived polypeptide derived precipitated by direct filtration split the mixture in cold methyl-tert-butyl ether (MTBE) and the precipitate was separated by centrifugation, washed twice with ether and air-dried.

Then the crude product is dissolved in dilute acetic acid, lyophilizers, pererastayut in 50% acetic acid and applied on a column with Sephadex G-50 (h cm)using 50% acetic acid as mobile phase. The collected fractions analyzed by MALDI-TOF mass spectrometry and those fractions which contain the desired polypeptide derivatives (MV 8436,9 Yes), unite and lyophilizers after dilution with water.

Combined fractions again dissolved in 50% acetic acid and further purified by drawing on prepreparation column Vydac C4size h mm Samples elute at the speed of a current of 3 ml/min using a linear gradient 20-80% B over 60 minutes, where In represents 0.1% of TFU in acetone the Rila mountain range, and represents 0.1% of TFU in the water. Detection is carried out at a wavelength of 280 nm, and only the fractions containing the target polypeptide are pooled and lyophilizers before carrying out further process of collapse.

Collapsing derived chemokine, purified by HPLC-OFF, carried out first by dissolving 10 mg of product in 1 ml of 6M GnHCl, 0,1M Na2HPO4and 10 mm Tris at pH 8.0 and at room temperature. After 20 minutes the solution is diluted by adding 10 ml of water to final concentrations of 0,6M GnHCl, 10mM Na2HPO4, 1mM Tris, pH 7.2 and the concentration of peptide 1 mg/ml Collapse initiated by the addition of cysteine at a concentration of approximately 20mm (approximately 100-fold molar excess relative to the concentration of peptide) and gradually increase the temperature to 37°C.

The reaction collapse occurring at a constant temperature of 37°s on the air track by analysis by HPLC OF using aliquot 25 ál of a solution, quenched with acetic acid, the instrument Waters 2690 Separation Module equipped with a photodiode detector Waters 996, using analytical column Vydac C4and 20-60% gradient of acetonitrile in the mixture of 0.1% TFU/water for 40 minutes at the speed of a current of 1.0 ml/min 1 µl of each HPLC peak (corresponding to the intermediate products of thiol-disulfide exchange rate is in place) collect, mixed with 1 μl of a saturated solution of sinoway acid in a mixture of acetonitrile/1% TFU (1:2) in water, dried in vacuum and analyzed by MALDI-TOF mass spectrometry using a spectrometer (Voyager-DE (Perseptive Biosystem, Framingham, MA)equipped with a nitrogen laser. 24 hours is formed 78% of the folded polypeptide. Peak MV which corresponds to the molecular weight of the folded product, then check in the reaction with N-ethylmaleimide (NEM) to detect the presence of free thiol groups (+125 Yes for each SH group).

Evaluation of biological activity of hu-TARC, obtained by the method according to the present invention, carried out according to the method of Imai (T. Imai et al., J. Biol. Chem., 271, 21514, 1996).

T-cell lines human Hut 78, Hut 102 and Jurkat, and fresh monocytes, neutrophils and lymphocytes assessed on their ability to migrate through the polycarbonate filter in response to the impact of TARC. In monocytes or neutrophils were observed chemotactic reaction either under the action of TARC, obtained by chemical synthesis, or under the action of the recombinant TARC. In the T-cell line Hut 78 and Hut 102 synthetic TARC, as well as recombinant TARC, induces migration of achieving a typical bell-shaped curve, and the maximum effect is observed at a concentration of 100 ng/ml.

Example 2

Synthesis and folding Cys10,34,50(S-t-Bu)-hu-TARC and

Cys11,34,50(t-Bu)-hu-TARC

Synthesis, purification and folding Cys derivatives10,34,50(S-t-Bu)-hu-TARC and Cys11,34,50(S-t-Bu)-hu-TARC is carried out in the same conditions that have been taken to Cys10,11,34,50(S-t-Bu)-hu-TARC (example 1), the only difference is that create Trt-protection for Cys10and Cys11respectively, which are removed simultaneously with the removal of polypeptide precursors from the resin. Outputs are minimized chemokines comprise 80% and 79%, respectively.

Example 3

Synthesis and folding Cys34,50(S-Bu)-hu-TARC

Synthesis, purification and folding derived Cys34,50(S-Bu)-hu-TARC is carried out in the same conditions that were used for derivatives in examples 1 and 2, except that both Cys residue10and Cys11protect Trt, which is removed during the final cleavage from the resin with TFU. The output of the folded product is 75%.

Example 4

Synthesis and folding Cys10,11,26,34,50,68(S-t-Bu)-hu-I-309

Synthesis of hu-I-309, containing 6 cysteine protected (S-t-Bu), carried out on a scale in increments of 0.12 mmol in the same conditions that were described in example 1, using Fmoc-Lys(Boc) - Wang resin (Wang resin) (degree of substitution is 0.61 mmol/g). The resulting complex polypeptide-resin is treated as described in example 1 and purified on G-50 material further purified by applying to the column Vydac C18size h mm (as the show is about to figures 1 and 2).

Collapsing derived chemokine, purified by HPLC OF conduct by dissolving 65 mg of the product in 60 ml of 0,6M GuHCl, 10mm Na2HPO4and 1 mm Tris at pH 8.0 and adding cysteine at concentrations up to 100-fold molar excess relative to the concentration of the peptide. The polypeptide solution is maintained at a temperature of 37°C for 4 days. After acidification with TFU rolled material produce by HPLC OF using column Vydac C18size h mm (as shown in figure 3 and 4). Completeness of coupling cysteine check using mass spectrometry after reaction with N-ethylmaleimide (NEM). No increase in mol. weight when this is not observed, indicating the absence of free thiol groups (as shown in figure 5). The output of the collapsed chemokine is almost 25%. Synthetic collapsed hu-I-309 demonstrates biological activity equivalent to the activity of the recombinant protein (6).

Conduct analytical chromatography was carried out using the following conditions:

Column: With18size h,6 mm (Vydac#238TP54)

Mobile phase: A = 100% H2O / 0.1% of TFU

In = 100% CH3CN / 0.1% of TFU

Gradient: composition In% described in the chromatogram

Detection: 214 nm

Example 5

The synthesis and folding of the C-terminal fragment

Plasmodium vivax

The synthesis of ochistka circumsporozoite protein Plasmodium vivax(PvCS 303-372), contains 4 cysteine-protected (S-t-Bu), carried out in the same conditions that were described in example 1.

Folding is conducted by adding 27 mg of peptide in 2.7 ml of 6M GuHCl in 0,1M Tris buffer pH 8.5. The resulting solution was stirred for 10 minutes. Then added to 13.5 ml of 1 mm EDTA, 0.2m NaCl, buffered at pH 8.8 in 0.2m Tris-buffer. In the end add to 10.8 ml 35mm cysteine in 1 mm EDTA, 0.2m NaCl, buffered at pH 8.8 in 0.2m Tris-buffer. The reaction mixture is brought to a temperature of 37°C. the Reaction clotting checked for completeness by HPLC with reversed phase (3-6 hours) (Fig.7) and stop the reaction by cooling for 5 minutes at 4°C, followed by addition of 10% TFU at 4°C to achieve a final concentration of 1% TFU (3 ml of 10% TFU). Subsequently, the product was then purified by HPLC with reversed phase (Fig) and determine the weight of the finished product (Fig.9). The final oxidized product is 70-80%.

Conduct analytical chromatography was carried out using the following conditions:

Column: With4size h,6 mm (Vydac#214TP54)

Mobile phase: A = 100% H2O / 0.1% of TFU

In = 100% CH3CN / 0.1% of TFU

Gradient: composition In% described in the chromatogram

Detection: 214 nm

Example 6

Large-scale synthesis and folding

C-terminal fragmentPlasmodium falciparum

KRU the scale synthesis and purification circumsporozoite protein Plasmodium falciparum(PfCS 282-383)containing only 2 cysteine-protected (S-t-Bu)4, is carried out in the same conditions that were described in example 1 (as shown in figure 10 and 11), except for the following changes.

Folding is conducted by dissolving 1.1 g of partially purified peptide in 1.0 l of 0.1 m CH3COONH4, pH 8.0 without the addition of free cysteine in the buffer to collapse. The reaction mixture was kept at a temperature of 32°C for 18 hours. The resulting product was then purified by HPLC with reversed phase (Fig and 13). The final oxidized product is nearly 37%.

Conduct analytical chromatography was carried out using the following conditions:

Column: With18size h,6 mm (Vydac#238TP54)

Mobile phase: A = 100% H2O / 0.1% of TFU

In = 100% CH3CN / 0.1% of TFU

Gradient: composition In% described in the chromatogram

Detection: 214 nm

1. The way spatial packaging (folding) chemically synthesized polypeptide, which includes the processing of the polypeptide and/or protein, which contains two or more S-butyl tolstonog residue, cysteine, in the buffer to collapse, which has an average pH value of from 7 to 9 and a temperature in the range from 25 to 40°C.

2. The method according to claim 1, characterized in that the specified buffer for minimizing includes one or more chaotropic Sol is th.

3. The method according to claim 2, characterized in that the said chaotrope salt selected from the group consisting of chloride guanidine and urea.

4. The method according to claim 2 or 3, characterized in that chaotrope salts are present in the buffer to collapse in a concentration of 0.1-1 M

5. The method according to any one of claims 1 to 4, characterized in that the specified buffer to minimize the has an alkaline pH value.

6. The method according to claim 5, characterized in that the pH value is from 7 to 8.5.

7. The method according to claim 1, characterized in that the temperature ranges from 27 to 38°C.

8. The method according to claim 7, characterized in that the temperature is approximately 37°C.

9. The method of obtaining biologically active peptide that includes

(a) chemical synthesis of the polypeptide that contains two or more S-butyl tolstonog balance;

(b) processing the specified polypeptide with cysteine in the buffer to collapse, which has an average pH value of from 7 to 9 and a temperature in the range from 25 to 40°; and

(c) clearance obtained folded polypeptides and/or proteins.

10. The method according to claim 9, characterized in that the buffer for minimizing includes one or more chaotropic salts.

11. The method according to claim 10, characterized in that chaotrope salt selected from the group consisting of chloride guanidine and urea.

12. The method according to claim 10, characterized in that chaotrope salt is rootstown in the buffer to collapse in a concentration of 0.1-1 M

13. The method according to any of PP-12, characterized in that the buffer for folding has an alkaline pH value.

14. The method according to claim 9, characterized in that the pH is from 7 to 8.5.

15. The method according to 9, characterized in that the temperature ranges from 27 to 38°C.

16. The method according to item 15, wherein the temperature is about 37°C.

17. The method according to any of p-16, includes stage

(a) Assembly S-tert-butyl-Tolstonogov polypeptide in an insoluble polymeric substrate through a step-by-step chain elongation;

(b) removal of the specified S-tert-butyl-tolstonog polypeptide chain from a specified substrate through acidolysis;

(c) purification of the obtained S-tert-butyl-Tolstonogov polypeptide;

(d) folding the purified S-tert-butyl-Tolstonogov polypeptide by processing the above polypeptide derived molar excess of cysteine in the buffer to minimize containing chaotrope salt and having an alkaline pH and a temperature of about 37°; and

(e) purification of the obtained folded proteins by high-performance liquid chromatography with reversed phase.

18. The method according to 17, characterized in that the specified jatropha salt is a chloride guanidine.

19. The method according to 17 or 18, characterized in that indicated the data of the polymer substrate is a polyamide or a resin based on polystyrene, functionalized cyclotourism linker in the form of hydroxymethyltransferase acid.



 

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