CZ80499A3 - Modification process of polypeptides - Google Patents

Abstract

A method of modifying polypeptides to facilitate their purification, which comprises inserting at least one specifically cleavable amino acid at the end of the polypeptide chain during its synthesis and protecting the same amino acid (s), if any, from cleavage to allow specific cleavage at the the site of a specifically cleavable amino acid (s). A method for producing purified polypeptides using said modification method.

Description

The present invention relates to a method of modification to facilitate the preparation of purified polypeptides and a method of purification utilizing this modification method.

BACKGROUND OF THE INVENTION

In recent years, solid phase peptide synthesis using the t-Boc or F-moc method has been greatly improved. Sophisticated synthesis protocols have allowed the preparation of polypeptides having approximately 100 or more amino acid residues (P. Chong, C. Sia, E. Tam, A. Kandil, M. Klein, International Journal of Peptide and Protein Research 41, 21-27, 1993; LR Haaheim, JP Maskell, P. Mascagni, AR Coates, Scandinavian Journal of Immunology 34, 341-350, 1991; MA Rogerro et al., Molecular Immunology 32, 1301-1309, 1995; DD Smith et al., International Journal of Peptide and Protein Research 44, 183-191, 1994. However, incomplete chain binding and termination, which can occur in any peptide elongation cycle, results in the formation of incomplete and truncated sequences.

This phenomenon and the possible occurrence of side reactions observed especially during the final cleavage from the resin complicates the direct isolation of the desired peptide from other impurities. The purification of long synthetic polypeptides is a major problem in the production of products suitable for biological studies and for use in humans and animals where a high level of purity is mandatory.

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Specifically, when sequences containing 30 or more amino acid residues are synthesized, the differences in physical properties such as size, charge and hydrophobicity of the desired product and the deleted sequence peptide may be too small after truncation or modification to allow sufficient purification. In addition, modern powerful separation techniques, such as reverse phase HPLC, are often limited by low yields and small sample injectable volumes, so they are lengthy and expensive.

Various approaches have been tested to circumvent this limitation. Biotinylation of residue 153 of the synthetic IL-1 protein (TJ Lobl, MJ Deibel, AW Yem, Analytical Biochemistry 170, 502-511, 1988) and residue 99 of the protease synthetic SIV protein (AG Tomasselli et al., Journal of Biological Chemistry 267) was performed. 10232-10237, 1992) and biotinylated chains were isolated on an avidin-agarose column.

Packaging by co-authors (HL Balí, P. Mascagni, International Journal of Peptide and Protein Research 40, 370-379, 1992) recently proposed a purification procedure based on the addition of a reversible protecting group providing lipophilic, acidic or basic functions to the last amino acid residue of the peptide chain .

More specific chromatographic methods have been optimized by utilizing the presence of special residues in the synthesized sequences. For example, cysteine containing peptides were purified by reaction with immobilized mercury derivatives (D.E. Krieger, B.W. Erickson, R. B. Merrifield, Proceedings of the National Academy of Sciences of the U.S.A 73, 3160-3164,

1976) or activated thiols (G. Lindeberg, J. Tengborn, H. bennich, UJ Ragnarsson, Chromatography 156, 366-369, 1978) and for purification of peptides containing histidine or tryptophan (G. Lindeberg, H. Bennich, A. Engstrom) Immobilized Metal Ion Affinity Chromatography (IMAC) has been used successfully (J. Porath, J. Carlsson, I. Olsson, G. Belfrage), and has been successfully used in the International Journal of Peptide and Protein Research 38, 253-259 (1991). , Nature 258, 598-599, 1975).

For recombinant proteins, histidine tails, B-cell epitope or GST particles were deliberately added. These ends could then be used in affinity chromatography.

In general, the purification protocol, which is based on the physicochemical properties of the synthesized peptide, must be optimized for each individual sample, which is a time-consuming and expensive procedure. For this reason, a number of techniques have been developed to allow the general applicability of purification procedures (G. Barany, R. B. Merrifield, Peptides, analysis, synthesis, biology 1-163-165, Academic Press, London 1980). However, the methods described so far are not entirely satisfactory as they are still time consuming and / or leave covalently derivatized peptides in the final purified products, which may be of some concern due to their biological and physicochemical properties and their end use in animals and humans.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to allow improvements

The known methods provide a method of modifying polypeptides to facilitate their purification and a process for purifying polypeptides, wherein the method and process are universally applicable, resulting in high yield and easy to carry out.

This object has been achieved according to the invention by a modification method which comprises inserting at least one specifically cleavable amino acid at the end of the polypeptide chain during its synthesis and protecting the same amino acid (s) in the polypeptide, if any, from cleavage to allow specific cleavage at the site of a specifically cleavable amino acid (s).

The purification procedure using this modification method comprises the steps of:

a) synthesizing the polypeptide of interest;

b) adding at least one specifically cleavable amino acid to the end of the polypeptides while protecting the same amino acid (s) in the polypeptide, if any, from cleavage;

c) extending the polypeptides and the amino acid (s) added thereto by a tag sequence to obtain the extended polypeptides;

d) purification of the elongated polypeptides using purification methods specific to the indicator sequence; and

e) removing the indicating sequence and the additional amino acid (s) from the extended polypeptide by a cleavage method specific to the added amino acid (s) to obtain a purified polypeptide.

• ·

The specific purification method is preferably a chemical cleavage method, as will be explained hereinafter.

The method and process of the invention are applicable to each polypeptide as they are not dependent on its amino acid composition. Furthermore, in certain preferred embodiments, the method and process of the invention are cost effective and highly efficient.

Such an embodiment is the use of a methionine residue as an additional amino acid prior to the addition of an affinity indicator sequence (e.g., a sequence of six histidine residues or other compounds to facilitate purification). After suitable purification steps, such as affinity chromatography specific to the indicator sequence, the histidine indicator sequence can be cleaved by digestion with CNBr, a cost-effective and highly efficient procedure that specifically cleaves at the site of the methionine residue.

Thus, in a preferred embodiment, the indicator sequence comprises at least a sequence of histidine residues, preferably six or more, and a methionine residue. Optionally, one or more other amino acids may be incorporated.

If the sequence of the polypeptide of interest contains methionine residues, they could be subject to CNBr cleavage when the indicator sequence is removed. However, according to the present invention, this can be avoided by using modified methionine residues in the synthesis of the polypeptide. Such a modified methionine residue is, for example, methionine sulfoxide.

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In an alternative embodiment of the method and process of the invention, the indicator sequence is a bulky molecule such as polyethylene glycol. In this case, the purification method specific to the indicator sequence is gel filtration chromatography.

The method and method of the invention are equally well applicable to polypeptides produced by recombinant DNA techniques. In a preferred embodiment, the methionine residues originally present in the desired polypeptide but not in the indicator sequence are protected against cleavage, for example by substitution with another amino acid such as valine or glycine, or are removed.

In this application, an indicator sequence is used to indicate a removable molecule added to a desired polypeptide during or after its synthesis. The indicator sequence may be an amino acid sequence added during polypeptide synthesis, but it may also be another molecule that can be readily purified from a mixture of components. An example of the latter is polyethylene glycol (PEG).

In this application, the terms peptide, and polypeptide are used interchangeably.

The following example is given to illustrate the invention. It will be appreciated by those skilled in the art that this example will provide sufficient guidance to develop other methods within the scope of the invention. An example is a generally applicable purification procedure based on a combination of 1) a capping protocol, 2) the use of methionine sulfoxide as a protected methionine residue during the synthesis of a native sequence, and 3) · • «· · extension of the desired peptide by methionine and two glycine residues and a final sequence of six histidines to be used for affinity chromatography. After sufficient purification steps, the histidine indication sequence was cleaved with cyanogen bromide (CNBr) followed by a final reduction of methionine sulfoxide to methionine. This simple, direct strategy allowed the purification of the 69 amino acid residue PbCS 242-310 polypeptide covering the C-terminal region of the CS protein of Plasmodia berghei to homogeneity, high yield, and in a short time using conventional chromatographic techniques.

Description of the drawings

Figure 1 shows the mass spectrum of the crude peptide.

Figure 2 shows the chromatographic profile of IMAC (immobilized metal ion affinity chromatography) purification.

Figure 3 shows the mass spectrum of the purified peptide.

Figure 4 shows the chromatographic profiles of the crude and purified peptide.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

1. Material and methods

1.1. Reagents and solvents

Chemicals and solvents used for peptide synthesis 0 0 00 0 0 0 0 0 0 0 0 0 0

0 0 0 0 0 · 00 0 000 • 0 · 0 · 0 0 were obtained from Calbiochem-Novabiochem AG (Laufelfingen, Switzerland) and Fluka (Buchs, Switzerland).

1.2. Peptide synthesis and analysis

To illustrate the present invention, a polypeptide, designated PbCS 242-310, covering the C-terminal region of the CS protein of Plasmodia berghei (DE Laran, Mol. Biochem. Parasitol. 39, 151-154, 1990) was chemically synthesized using the F-Moc method. solid phase on an Applied Biosystems 431A peptide synthesizer. The polypeptide was prepared on F-moc-Ser (t-butyl) -p-alkoxybenzyl alcohol resin (Wang resin) with a degree of substitution of 0.43 mmol / g on a 0.1 mmol scale. Synthesis was performed using a 5-fold excess of amino acid derivatives of F-moc, DDCI and HOBt as activating agents, a 60 minute binding time for the first 34 amino acids, and a double binding time for the remaining residues. Capping with acetic anhydride was performed at the end of each cycle. Side chain protecting groups included: a pentamethylchromansulfonyl group for Arg; -S-t-butyl for Cys; a triphenylmethyl group for Asn, Gln and His; t-butoxycarbonyl for Lys and Trp; t-butyl for Asp, Clu, Ser, Thr, and Tyr. Met 306 was inserted as Fmoc-Met-sulfoxide to protect against later cleavage of CNBr.

Then, the peptide was extended N-terminally with the His-His-His-His-His-His-His-Gly-Gly-Met sequence under the same conditions as described above, but the capping was omitted after the second Gly was bound. The polypeptide thus obtained is referred to as His tag PbCS 242-310.

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The crude peptide was obtained by treating the peptide-resin system with 2.5% H ₂ O, 5% triethylsilane in TFA (trifluoroacetic acid) for 2 hours at room temperature. The synthetic peptide was purified by liquid chromatography on a 70 x 2.5 cm Sephadex G-50 column using 50% acetic acid / H ₂ O as the mobile phase. Purity of the peptide was analyzed by reverse phase HPLC using a C4 W-Porex 250 x 4.6 mm column and a 10-90% CH ₃ CN gradient in 0.1% TFA / H ₂ O over 60 minutes and a flow rate of 1.0 ml / min. The amino acid composition was determined by Knecht and Chang (R. Knecht, JY Chang, Analyt. Chem. 58, 2375-2379,

1986).

1.3. Immobilized metal ion affinity chromatography (IMAC) and CNBr cleavage

The polypeptide was first purified by affinity chromatography based on a histidine indicator group. Later, the indicator group was removed by CNBr.

Ni-column (Quiagen Inc., Chatsworth, USA) was prepared with Ni-NTA agarose resin and equilibrated with buffer A (8 Mol. ^-1 I urea, 0.1 mol. I ^'1 Na2HPO4, 0.01 mol.l ¹ Tris, pH adjusted to 8.0 with H ₃ PO ₄ ). The gel-purified His tag PbCS 242-310 polypeptide was dissolved in buffer A and loaded onto the column at a flow rate of 15 ml / hour. The column was washed with buffer A (flow rate 15 ml / h) and buffer B (8 mol.l ^-1 urea, 0.1 mol.l ¹ Na ² HPO 4, 0.01 mol.l ¹ Tris, pH adjusted to 6.3 by H ₃ PO ₄ ) containing 50 mmol. ¹ imidazole at a flow rate of 30 ml / hour. The His-tag PbCS 242-310 was then eluted (30 ml / hr flow rate). 9 9

9999999 · 9 99 99 buffer B containing 250 mmol.l- ¹ imidazole.

The eluted material was desalted on a Sephadex G25 column (50 x 2.5 cm, using 50% acetic acid / H ₂ O as the mobile phase) and lyophilized. To remove the histidine indicator sequence, the material so obtained was treated for 8 hours on the reverse phase at a concentration of 20 mg / ml in 70% TFA using a 100-fold molar excess of CNBr.

The digested material was lyophilized, dissolved in buffer A, and loaded onto a Ni-NTA agarose column. The histidine indicator sequence is captured on the Ni-column and the eluent from the column contains the PbCS 242-310 polypeptide. The eluent from the column was desalted on a Sephadex G25 column (50 x 2.5 cm, using 50% acetic acid / H ₂ O as the mobile phase) and lyophilized.

1.4. Reduction of Met-sulfoxide

CNBr-treated material and purified IMAC at pH 8.0 were treated with 10% mercaptoethanol to convert methionine sulfoxide to methionine and Cys-S-t-butyl to Cys and then purified by gel filtration (Sephadex G25 column, 250 x 4.4 mm).

1.5. Mass spectrometry

Mass spectrographic analysis was performed using an LDI 1700 Mass Monitor fly-through mass spectrometer (Linear Scientific Inc, Reno, NV, USA) to evaluate flight time. Five microliters of 1 mg / ml polypeptide solution was mixed with 5 μΐ · 9 · 9 · 9 · 9 · 9 · 9 Φ Φ φ φ

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Trans trans trans-3,5-dimethoxy-4-hydroxy-cinnamic acid (sinapinic acid) at 20 mg / ml in acetonitrile (Linear Scientific Inc, Reno, NV, USA) and 1 μΐ of this mixture was applied to the tip of the probe mass spectrometer and dried under gentle vacuum. The sample was irradiated with 3-ns laser pulses (wavelength 337 nm) from the N ₂ -laser. The flyby time was measured with a digital oscilloscope (9304 series; Le Croy Research Systems, Corp., Spring Valley, NY) which was converted to mass spectra using the Peptide MALDITOFMS Calibration Standard (Linear Scientific Inc., Reno, NV, USA).

2. Results

PbCS 242-310 with 69 amino acid residues corresponds to the C-terminal region of the CS protein of P. berghei (D.E. Lanař, Mol. Biochem. Parasitol. 39, 151-154, 1990).

Synthesis of His-tagPbCS242-310 was performed using an automated procedure protocol in which a capping step was incorporated after each binding as described in the Materials and Methods section.

Treatment of 600 mg of the corresponding peptide resin with H ₂ O / triethylsilane / TFA gave more than 150 mg of crude polypeptide.

Mass spectral analysis of the crude polypeptide showed the presence of the desired substance with a molecular weight (MW) of 9301 among other low molecular weight components (FIG. 1).

mg of crude polypeptide was purified by affinity ·

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Immobilized metal ion chromatography (IMAC) on a 25 ml Ni-NTA agarose column (FIG. 2). After desalting by liquid chromatography separating substances of a certain molecular size, 35 mg of His-tag PbCS242-310 were obtained. Measurement of the absorbance of the eluted material (35 mg) at 280 nm and all flow (55 mg) showed that the purification yield was 100%.

The material purified on a Ni-column was then digested with CNBr to remove the six His residue indicator sequence.

The digestion material was then re-deposited on a Ni-column to remove uncleaved peptide and exposed to 10% mercaptoethanol at pH 8.0 to reduce the methionine sulfoxide introduced during the synthesis and the cysteine residue protecting groups. Complete reduction of methionine sulfoxide was confirmed by re-exposure to CNBr and verification of cleavage efficiency by mass spectrography.

Further purification by size exclusion chromatography yielded 19 mg of purified PbCS 242-310.

Figure 3 shows the mass spectrum obtained by comparing the analytically purified peptide. The runs are due to the material and Figure 4 shows the chromatographic profiles of the crude and the difference in retention times of the two absence of a highly charged histidine indicator group in the purified material. Purified PbCS 242-310 was found to exhibit about 95% purity by integrating peak areas (peaks) in a 214 nm wavelength analysis. The amino acid composition of the CS polypeptide was consistent with

0 0 0 0 0 0 · 0 0 · 0 0 0 0 0 0 0 00000000 · · the composition predicted for this peptide (Table 1)

Table 1: Amino Acid Analysis

amino acid ³ residues per mole of PbCS 242-310 awaited observed ^b SD ^C Asp + Asn 10 9.4 1.0 Glu + Gln 9 9.1 0.9 Ser 7 8.4 2.1 Thr 4 2.2 WHAT O Gly 3 3.1 1.0 Ala 2 3.4 1.1 Arg 4 4.8 0.9 For 1 1.1 0.2 Wall 3 2.9 0, 3 Met 1 1.0 0.1 Ile 7 6, 3 0.7 Leu 3 3.3 0.2 Phe 1 1.1 0.1 Lys 8 6.7 1.0 His 0 - - Tyr 1 1,2 0.2

^and Cys and Trp were not determined ^{b the} mean of five determinations ^c standard deviation

3. Discussion

Chemical synthesis of bioactive peptides has become a widespread and rapidly expanding technique due to automation and efficient chain assembly procedures. For most applications, the raw synthetic product must be purified to remove remaining reagents, missing sequences, and chemically modified peptide types. This is usually done by reverse phase HPLC using a system.

- ± 4 ~ · ** ··· 9 9 9 9 9 999999 9 9 9 9 9 9999 99 9999 99 aqueous trifluoroacetic acid / acetonitrile as the mobile phase. Although peptide synthesis has become highly automated, purification is still predominantly a manual process and thus a time consuming, costly, and not very efficient process.

Example 1 has shown that the process of the invention results in high purity as shown in Figure 3 and is easy to perform and universally applicable.

The problem of specific cleavage due to the presence of methionine residues in the desired sequence, as in the example herein, can be easily overcome by the use of CNBr resistant methionine sulfoxide residues quantitatively reducible by mercaptoacetic acid (RA Houghten, CH Li, Methods in Enzymology 91, 549-559 (1991).

It follows from the above that the process of the invention was successfully used to purify the PbCS 242-310 ”polypeptide, a chain of 69 amino acid residues corresponding to the C-terminal region of the CS protein of P. berghei (ref. 12). Although many peptide impurities were present in the raw material after cleavage from the resin, as shown by the mass spectral analysis presented in Figure 1, the inventors were able to purify the target peptide to homogeneity with high yield and in a relatively short time. A complete purification protocol yielded 19 mg of purified PbCS 242-310, corresponding to approximately 20% of the crude material.

In conclusion, it has been shown that the cleavage of CNBR and the protection of the corresponding methionine residues as sulfoxides in conjunction with an effective affinity indicator sequence may be effective.

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Claims (7)

PATENT CLAIMS A method of modifying polypeptides to facilitate purification thereof, the method comprising modifying at least one specifically cleavable methionine and an identification sequence at the end of a polypeptide chain during its synthesis and protecting methionine residues in the polypeptide, if present, from cleavage by sulfoxide protection, wherein the indicator sequence is a histidine identification sequence or a larger molecule such as polyethylene glycol. A method for producing purified polypeptides using the modification method of claim 1, wherein the method comprises the steps of: a) synthesizing the polypeptide of interest; b) adding at least one specifically cleavable methionine to the end of the polypeptide while protecting the methionine (s) in the polypeptide, if any, from cleavage by sulfoxide protection; c) elongating the polypeptide and the methionine (s) added thereto by an indicator sequence to obtain the elongated polypeptide; d) purifying the elongated polypeptide by a purification method specific to the indicator sequence; and e) removing the indicating sequence and the additional methionine (s) from the elongated polypeptide by a cleavage method based on the use of cyanogen bromide to obtain a purified polypeptide, characterized in that the indicating sequence is a histidine indicator sequence or a larger molecule such as polyethylene glycol. · · · • φ φ φ φ φ φ φ φ φ φ φ φ The method of claim 2, wherein the histidine indicator sequence consists of His-His-His-His-His-His-His-Gly-Gly-. Method according to claim 2 or 3, characterized in that the purification method specific for the indicator sequence is chromatography based on affinity for the indicator sequence. The method of claim 2, wherein the indicator sequence is bulk polyethylene glycol and the method of purification, the indicator sequence, is gel filtration. a growing molecule such as specific for polypeptide recombination Method according to claims 2 to 5, characterized in that the polypeptide of interest is produced by DNA techniques in a living host and the methionines v are removed or replaced by another amino acid, such as zalin, glycine or modified methionine, instead of being protected by a sulfoxide group. The method of claim 6, wherein the living host is a eukaryotic host or a prokaryotic host, such as Escherichia coli.