Getting treated (poly)peptides

 

(57) Abstract:

Describes how modifications(poly)peptides comprising embedding at least one specific split methionine and tag (tag), which is a His-tag at the end of the (poly)peptide chain during synthesis and protection of methionine residues in this (poly)peptide, cleavage by protecting their sulfoxide group, and also describes a method of obtaining purified (poly)peptides, providing stages: a) synthesis of the target peptide; b) attaching at least one specific split methionine at the end of this (poly)peptide, while methionine (methionine) in the (poly)peptide are protected against cleavage by protecting sulfoxide group) elongation (poly)peptide and methionine(methionine) attached to it, the label with getting the elongated polypeptide, g) cleaning elongated (poly)peptide using the cleaning method, specific to the label, d) removal of the label and additional methionine (methionine) of the elongated (poly)polypeptide by cleavage of cyanocinnamic, receiving cleared (poly)peptide, moreover, the tag is a His-sequence Kăđẫa getting treated (poly)peptides and purification method using this modification.

Art

In recent years, solid-phase peptide synthesis using t-Boc - and F-moc strategy was largely improved. Sophisticated protocols synthesis allowed us to obtain polypeptides of about 100 or more residues [1-4]. However, incomplete coupling and termination circuit, which can occur in any cycle of peptide Assembly leads to the formation of deletions and truncated sequences.

This and the possible presence of adverse reactions observed mainly during the final cleavage from the resin, complicate direct selection of the desired peptide from other impurities. Cleaning long synthetic polypeptides is the main problem in obtaining products which are applicable for biological research and for use for humans and animals, where a high level of purity is required.

in particular, when synthesized sequence containing 30 or more residue differences in physical properties, such as size, charge and hydrophobicity between the desired product and deleteregvalue, truncated or modified peptide impurities may be too small to allow adequate clearance. Cu is their exits and small bootability samples that takes a lot of time and is expensive.

Different approaches were tested to overcome this limitation. Were done biotinylation synthetic protein IL-1 [5] of 153 residues and synthetic protein SIV protease [6] of 99 residues and biotinylated chains were isolated by avidin-agarose column.

Ball et al. [7] recently proposed a purification procedure based on the accession of the reversible protective groups, lipophilic carrier, acidic or basic functional group, to the last residue of the peptide chain.

Were optimized more specific chromatographic methods, exploiting the presence of certain residues in synthetic sequences. For example, cysteinaemia peptides were purified by reaction with immobilized derivatives of mercury [8] or activated thiols [9] and was successfully applied to affinity chromatography with immobilized metal ion (IMAC) [10] for the purification of peptides containing histidine or tryptophan [11].

In recombinant proteins were purposefully attached his-tag tail, b-cell epitope or GST-part of the molecule. These tails could be used in affine chromatog peptide, should be optimized for each individual sample, which is long and costly. For these reasons, it was developed a number of ways to make the cleaning procedures General applicability [15]. However, the methods described so far, are completely unsatisfactory and/or leave covalently derivationally peptides in the final purified products, which can create some anxiety in relation to their biological and physico-chemical properties and their end use in animals and humans.

The invention

Thus, the purpose of this invention is the provision of improvements to known methods to create modification (poly)peptide to facilitate their purification and purification method (poly)peptides, which are universally applicable, leading to high yields of extraction, and simple to execute.

This goal was achieved in accordance with this invention by way of modification, which includes embedding at least one specifically cleaved amino acids at the end of this (poly)peptide chain during synthesis and protection of the same amino acids (amino acids) in the (poly)saleemi amino acid (amino acids).

The cleaning process using this modification provides for the stage:

a) synthesis of the desired (poly)peptide;

b) attaching at least one specifically cleaved amino acids at the end of this (poly)peptide in the protection of the same amino acids (amino acids) inside the (poly)peptide, if present, from splitting;

b) lengthening (poly)peptide and attached amino acids (amino acid) sequence-tagged for receiving the elongated polypeptide;

c) purification of the elongated polypeptide in through a purification method that is specific to the tag, and

d) removal sequence tags and additional amino acids (amino acids) of the elongated (poly)peptide by way of cleavage, specific additional amino acids (amino acids), with purified polypeptide.

Method-specific cleavage is a preferred method of chemical cleavage, as will be further explained here below.

The method and process of the present invention is applicable to any (poly)peptide, as they do not depend on its amino acid composition. In addition, in some preferred embodiments, isusulat the application of the methionine residue as additional amino acids before attaching affinity tag (tag) (e.g., cut six histidinemia residues, or other easy-to-clean joints). After appropriate stages of purification, such as a specific tag affinity chromatography his-tag label can be split by the splitting using CNBr, inexpensive and very effective way, which specifically cleaves at methionine residue.

In a preferred embodiment, the label (tag) contains, therefore, at least a segment of a his-tag residues, preferably six or more, and the residue is methionine. Optional and may be included with one or more other amino acids.

If the sequence of the desired polypeptide contains residues of methionine, they could be subject to cleavage by cyanogenmod when the label is removed. However, in accordance with this invention this can be avoided by the use of modified residues methionine in the synthesis of (poly)peptide. Such modified methionine residue is, for example, methanesulfonic.

In an alternative embodiment, the method and process of this invention the label is a large molecule, such as polyethylene glycol. In this case, specific to the label sparano well applicable to polypeptide, produced by means of recombinant DNA. In a preferred embodiment, residues methionine, originally present in the desired polypeptide, but not in the label (tag), are protected from cleavage, for example, being replaced by another amino acid, such as valine, glycine, or deleteregvalue.

In this application, the term "tag" (tag) is used to indicate the deleted molecule attached to the target polypeptide during synthesis or after synthesis. "Label" may be amino acid sequence attached during synthesis of the polypeptide, but may also be another molecule, which can easily be purified from a mixture of components. An example of the latter is polyethylene glycol (PEG).

In this application, the terms "peptide", "polypeptide" and "(poly) peptide" are used interchangeably.

Information confirming the possibility of carrying out the invention.

The following example is provided to illustrate the present invention. It is clear that the qualification specialist in this field, this example will provide a reasonable recommendation for the development of further methods, which are also included in the scope of this invention. In the example described, the cleaning procedure is methanesulfonamide as protected methionine residue and 3) elongation (extension) of the desired peptide segment from 6 histidines, which will be used for affinity chromatography. After adequate stages of purification was performed by splitting cyanogenmod his-tag labels with subsequent final restoration methanesulfonamide to methionine. This simple, direct strategy has made possible the purification to homogeneity of the polypeptide of 69 residues "PbCS 242-310" covering end area of the CS protein of Plasmodium berghei, with high yield and in a short time using conventional chromatographic procedures.

Example

1. Materials and methods

1.1 Reagents and solvents

Chemical reagents and solvents used for peptide synthesis were purchased from Calbiochem-Novabiochem AG (Laufelfingen, Switzerland) and Fluka (Buchs, Switzerland).

1.2. Synthesis and analysis of peptides

For illustration of this invention the polypeptide labeled "PbCS 242-310" covering end area of the CS protein of Plasmodium berghei [12], chemically synthesized using F-Moc chemistry solid phase peptide synthesizer A Applied Biosystems. This polypeptide were obtained on the resin with F-moc-Ser (tert-butyl)-p-alkoxybenzyl alcohol (Wang resin) with a degree of substitution of 0.43 mmol/g at 0.1 mmol scale. The synthesis was performed using 5-fold excess F-CBT-amino acid derivatives, DCCI and H is ia for the following residues. Blocking ("copy") of acetic anhydride was performed at the end of each cycle. Protective groups of the side chains included: pentamethylbenzenesulfonyl group for Arg; -S-tert-butyl CPS; triphenylmethyl group for Asn, Gin, and His; tert-butoxycarbonyl group for Lys and Tight; tert-boutelou group of Asp, Glu, Ser, Thr and Tight. Met 306 was built in the form of Fmoc-Met-sulfoxide to protect it from later splitting cyanogenmod.

Then the peptide was extended at the N-end of the sequence His-His-His-His-His-His-Gly-Gly-Met using the conditions described above, but did not "kupirovaniya" after binding of the second Gly. Thus obtained polypeptide was labeled "His tag PbCS 242-310" (where the tag refers to a tag).

The crude peptide was obtained by processing containing the peptide resin of 2.5% H2O, 5% triethylsilane in TN for 2 hours at room temperature. The synthetic peptide was purified by gel-filtration liquid chromatography (column Sephadex G50 702,5 cm using a mixture of 50% acetic acid/N2About as mobile phase). The purity of the peptide was analyzed OFF-VIH using column C4 W-Porex 2504,6 mm, gradient 10-90% CH3JV in a mixture of 0.1% of TN/N2About within 60 minutes, with a flow rate of 1.0 ml/mnum metal ion (IMAC) and splitting using CNBr.

The first polypeptide was purified by affinity chromatography on the basis of his-tag labels. Then the label was removed by cyanogenmod.

Ni-column was prepared with Ni-NTA-agarose resin (Qiagen Inc., Chatsworth, USA) and were balanced with buffer A (8M urea, 0.1 M Na2HPO4, 0.01 M Tris, pH, brought to 8.0 by using H3RHO4). Purified by gel-filtration polypeptide "His tag PbCS 242-310" was dissolved in buffer a and applied to the column with a flow rate of 15 ml/hour, the Column was washed with buffer a (flow rate 15 ml/h) and buffer B (8 M urea, 0.1 M Na2HPO4, 0.01 M Tris, pH, increased to 6.3 using H3RHO4) containing 50 mm imidazole, at a speed of DC 30 ml/hour. Then polypeptide "His tag PbCS 242-310 was suirable (flow rate 30 ml/hour) buffer containing 250 mm imidazole.

Suirvey material was absoluely using column Sephadex G25 (502,5 cm using a mixture of 50% acetic acid/N2About as mobile phase) and liofilizirovanny. To remove the his-tag labels thus obtained material was treated for 8 hours at room temperature at a concentration of 20 mg/ml in 70% of TN using 100-fold molar excess of CNBr.

Split the material thus liofilizirovanny, is olence and flowing through the column a solution containing polypeptide "PbCS 242-310". Flowing through the column, the solution was absoluely using column Sephadex G25 (502,5 cm using a mixture of 50% acetic acid/H2O as mobile phase) and liofilizirovanny.

1.4. Recovery of Met-sulfoxide

Processed CNBr and IMAC purified material was treated with 10% mercaptoethanol at pH 8.0 for turning methanesulfonamide in methionine and Cys-S-tert-butyl in Cys and then further purified by gel-filtration (column Sephadex G25 2504,4 mm).

1.5. Mass spectrometry

Mass spectrometric analysis was performed using time-of-flight mass spectrometer LDI 1700 Mass Monitor (Linear Scientific Inc., Reno, NV, USA). Five μl of solution 1 mg/ml of the polypeptide was mixed with 5 μl of TRANS-3,5-dimethoxy-4-hydroxyanisol acid (sinap(Jn)OIC acid)) (20 mg/ml in acetonitrile (Linear Scientific Inc. , Reno, NV, USA) and 1.0 μl of this solution was placed on the tip of the probe mass spectrometer and dried by gentle vacuum. The sample was illuminated 3-NS laser pulses (wavelength 337 nm) of N2-laser. The time of flight was measured with a digital oscilloscope (series 9304; Le Croy Research Systems, Corp. , Spring Valley, NY), which was turned into a mass spectrum using the calibration standard Peptide MALDI-TOFMS (Linear Scientific Inc., Reno, NV, USA).

2. Results

Polypeptide and the automated Protocol, in which stage of "kupirovaniya included after each binding, as described in "Materials and methods".

More than 150 mg of the crude polypeptide was obtained by treatment of 600 mg of the corresponding peptide resin with a mixture of N2About/triethylsilane/TN.

Mass spectral analysis of the crude polypeptide showed the presence of interest molecular particles with molecular weight (MW) 9301 among other components with low molecular weight (MW)(Fig.1).

90 mg of the crude polypeptide was purified by affinity chromatography with immobilized metal ion (IMAC) column with a volume of 25 ml of Ni-NTA-agarose (Fig. 2). After desalting by gel filtration liquid chromatography was obtained 35 mg "His tag PbCS 242-310". The absorption measurement at 280 nm lirovannomu material (35 mg) and passed through a column of solution (55 mg) showed that the yield of this purification Protocol was 100%.

Then purified on Ni-column material were digested CNBr for elimination 6IS tags.

Split the material repeatedly inflicted on Ni-column for the elimination of undigested peptide and treated with 10% mercaptoethanol at pH 8.0 to restore methanesulfonamide, built during sinteco material CNBr and checking the efficiency of the cleavage mass spectrometry.

Additional cleaning gel filtrational chromatography led to 19 mg purified PbCS 242-310.

In Fig. 3 shows the mass spectrum of the material obtained, and Fig.4 compares the analytical chromatographic profiles of the crude and purified peptide. Differences in retention time between the two divisions caused by the lack of vysokozaraznoy His label in purified material. It was found that purified "PbCS 242-310" has a purity of about 95%, based on the integration of the peak areas in the analysis at 214 nm. The amino acid composition of the polypeptide CS is consistent with the composition expected for this peptide (table).

Chemical synthesis of bioactive peptides has become widespread and rapidly growing method for automation and effective protocols to build circuits. For most applications the crude synthetic product should be cleaned to remove residual reagents failed sequences and chemically modified peptide molecules. This is usually accomplished by reversed-phase VIH using mobile phases triperoxonane acid/acetonitrile. Although peptide synthesis has become highly automated, cleaning is still very stephenstephen.

This example showed that the method of this invention results in high purity, as it follows from Fig.3, and is easy to perform and universally applicable.

The specific problem of splitting due to the presence of Met residues in the target sequence, as described in this case, in accordance with this invention is easily overcome by the use of Met-sulfoxide residues that are resistant to treatment by CNBr and quantitatively recovered mercaptohexanol acid [14].

From the above it follows that the method of the present invention was successfully applied for the purification of the polypeptide "PbCS 242-310" chains 69 residue corresponding to the C-terminal region of the protein CS P. berghei [12]. Although in the raw material after removal from the column contains many of peptide impurities, as shown by mass spectral analysis shown in Fig.1, the inventors were able to purify the target peptide to homogeneity with a high yield in a relatively short time. The full Protocol of purification gave 19 mg of purified bS 242-310, which corresponds to about 20% of the crude material.

In conclusion, it was demonstrated that the CNBr cleavage and protection of relevant OST wny and General cleaning tool chemically synthesized long-chain peptides.

Figure captions

Fig.1 - mass spectrum of the crude peptide

Fig.2 - chromatographic profile IMAC-purification

Fig.3 - mass spectrum of the purified peptide

Fig.4 - chromatographic profiles of the crude and purified peptide.

Sources of information

1. Chong, P., Sia, C., Tam, E., Kandil, A. & Klein, M. International Journal of Peptide &Protein Research 41, 21-27 (1993).

2. Haaheim, L. R., Maskell, J. P., Mascagni, P. & Coates, A. R. Scandinavian Journal of Immunology 34, 341-350 (1991).

3. Roggero, M. A., et al. Molecular Immunology 32, 1301-1309 (1995).

4. Smith, D. D., et al. International Journal of Pep-tide & Protein Research 44, 183 to 191 (1994).

5. Lobl, T. J. , Deibel, M. J. & Yem, A. W. Analytical Biochemistry 170, 502-511 (1988).

6. Tomasselli, A. G. , et al. Journal of Biological Chemistry 267, 10232-10237 (1992).

7. Ball, H. L. & Mascagni, P. International Journal of Peptide &Protein Research 40, 370-379 (1992).

8. Krieger, D. E., Erickson, B. W. & Merrifield, R. B. Proceedings of the National Academy of Sciences of the United States of America 73, 3160-3164 (1976).

9. Lindeberg, G., Tengborn, J., Bennich, H. & Ragnarsson, U. J. Chromatography 156, 366-369 (1978).

10. Porath, J. , Carlsson, J., Olsson, I. & Belfrage, G. Nature 258, 598-599 (1975).

11. Lindeberg, G., Bennich, H. & Engstrom, A. International Journal of Peptide &Protein Research 38, 253-259 (1991).

12. Lanar, D. E. Mol. Biochem. Parasitol. 39, 151-154 (1990).

13. Knecht, R. & Chang, J. Y. Analyt. Chem. 58, 2375-2379 (1986).

14. Houghten, R. A. & Li, C. H. Methods in enzymology 91, 549-559 (1991).

2. A method of obtaining purified (poly)peptides using modification under item 1, providing for the stage: a) the synthesis of the desired (poly)peptide; (b) attaching at least one specific split methionine at the end of this (poly)peptide, while methionine (methionine) in the (poly)peptide, if present, are protected against cleavage by protecting sulfoxide group; (c) elongation (poly)peptide and methionine (methionine) attached to it a label with getting the elongated polypeptide; (d) cleaning elongated (poly)peptide using the cleaning method, specific to the label; (e) removal of the label and additional methionine (methionine) of the elongated (poly)peptide using the method of splitting based on cyanogenmod, receiving cleared (poly)peptide, wherein the label is a-His-His-Gly-Gly.

4. The method according to p. 2 or 3, where the method of treatment specific to the label, is a chromatography based on affinity in terms of sequence tags.

5. The method according to PP. 2-4, where the target (poly)peptide is produced by means of recombinant DNA in a living host and instead of protecting sulfoxide group methionine in the (poly)peptide delegated or replaced by another amino acid, such as valine, glycine or modified methionine.

6. The method according to p. 5, where the living host is a eukaryotic host or prokaryotic host, such as Escherichia coli.

Priority points:

09.09.1996 - PP. 1-4;

02.05.1997 - PP. 5 and 6.

 

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