JPS6244920B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a protein, which comprises a protein containing a tetrapeptide sequence Pro-Xyz-Gly-Pro in which Xyz may represent any arbitrary amino acid, and the tetrapeptide sequence Pro-Xyz-Gly-Pro in any position. in the peptide sequence
It is characterized in that the Xyz-Gly bond is selectively cleaved by collagenase, and then the glycine residue is removed using aminoacylproline-aminopeptidase and the proline residue is removed using proline aminopeptidase.

When a heterologous protein (e.g. mammalian proteohormone) is synthesized by a genetically mutated microorganism,
At the 3' end of the codogener string, a heterologous gene containing one or more stop codons and coding for the desired protein sequence is incorporated into the structural gene of the microorganism. This allows the regulatory sites on the microbial DNA to still function and protein biosynthesis to proceed normally. As the first product of protein synthesis, a fusion protein is obtained which contains a somewhat longer sequence of the microbial protein at the amino terminus and the desired heterologous protein at the carboxyl terminus. The heterologous protein must first be cleaved from this fusion protein by a protein chemical reaction. The only method currently known for this cleavage is reaction with bromcyan, which causes cleavage of the peptide sequence at the carboxyl-terminal side of the methionine residue [SBNeedleman, et al.
“Protein Sequence Determination”, (1970
), published by Springer Verlag (New York)]. At that time, the foreign gene is transferred to the 5′-
It is necessary to contain an additional codon for methionine on the terminal side. However, this method is of no use if additional methionine residues occur in the desired heterologous protein. Furthermore, bromcyanic cleavage has the disadvantage of producing side reactions with various other amino acids.

By the way, if a tetrapeptide with the general formula: Pro-Xyz-Gly-Pro (wherein Xyz may be any amino acid) is present at the amino terminal side of the heterologous protein sequence in the fusion protein, normal side reactions may occur. Without,
It has been found that heterologous proteins can be obtained by selective enzymatic hydrolysis even if methionine residues are still present. First, the Xyz-Gly-bond is selectively cleaved with collagenase (EC3.4.24.3.) and the glycine residue is subsequently cleaved with aminoacylproline-aminopeptidase (aminopeptidase-P,E).
C.3.4.11.9.) and proline residues are removed with proline-aminopeptidase (EC3.4.11.5.).

Therefore, in the method of the present invention, the general formula: Pro-Xyz-Gly
Collagenase specificity of -Pro consists of specific enzymatic cleavage of a genetically engineered fusion protein with a tetrapeptide sequence, in which the enzymatic cleavage involves the enzyme collagenase, aminoacylproline-
Aminopeptidase and proline-aminopeptidase are used.

Xyz are all naturally occurring amino acids that are included in the genetic code.

The collagenase necessary for enzymatic cleavage of the desired protein from the fusion protein must be substantially free of other proteases. For example, this enzyme is produced from Clostridium histolyticum and can be obtained by conventional fermentation using known methods. It is also commercially available in purified form. To sequester proteases, which are still included in small amounts in most formulations, protease inhibitors, such as diisopropylfluorophosphate, are added during incubation with the fusion protein. Collagenase itself is not inhibited by this inhibitor. This reaction results in the desired heterologous protein extending amino-terminally towards the sequence Gly-Pro. It can be separated from the reaction mixture. The method used for this purpose essentially depends on the specificity of the compound, which is determined by the heterologous protein of interest, for example proinsulin, insulin A chain or insulin B chain, ACTH, STH.

Cleavage by specific aminopeptidases of both glycine and proline residues can also be achieved by performing them one after the other in two separate hydrolysis batches or, in many cases, by incubating both enzymes together. can do. Aminoacylproline-aminopeptidase can be isolated from E. coli strains, such as strain B, by ammonium sulfate fractionation, acetone precipitation and chromatography on calcium phosphate, DEAE-cellulose and Sephadex G-200.
[“Biochem.and Biophys.Res.Comm.” Volume 32,
See pages 658-663 (1968)]. Proline aminopeptidase is likewise isolated from E. coli strains, such as strain K12. It is obtained in sufficiently pure form after ammonium sulfate fractionation and two chromatographies on DEAE-cellulose. It must not contain aminopeptidases with low specificity ["J. Biol. Chem.", Vol. 294, pp. 1740-1746 (1959);
Year)〕. Furthermore, after the Xyz-Gly-bond of the N-terminal tetrapeptide sequence of the fusion protein is cleaved with collagenase, the dipeptide sequence Gly-Pro present at the amino terminus of the heterologous protein sequence is cleaved using post-proline dipeptidyl aminopeptidase in one step. It was found that it could be cleaved. This post-proline dipeptidyl aminopeptidase is readily available according to its description in the literature [“Biochim.Bio-phys.
Acta”, vol. 485, p. 391 (1977)].

The advantages of using this enzyme are the high activity of dipeptidyl aminopeptidase and the easy work-up of the final product. High enzymatic activity allows a very rational use of this method. This activity is maintained upon attachment of the enzyme to a carrier, which greatly simplifies the method. Because the cleavage of both amino acids Gly and Pro occurs in one step, the final product is derived from one by-product, rather than two by-products (the starting material and the amino acid truncated starting material) due to incomplete cleavage. Only the starting material needs to be separated in the final purification. However, this method is not applicable if the desired heterologous protein sequence begins with proline or aminoacylproline. i.e. PPDA (post-proline dipeptidyl aminopeptidase)
is Gly-Pro only if the dipeptide following it C-terminally does not contain proline or hydroxyproline in the first and second positions.
Cleave Pro from Gly-Pro-protein.

A portion of a fusion protein having a tetrapeptide sequence and a desired protein (referred to as protein sequence in this application) is illustrated as follows: By the way, the tetrapeptide sequence Pro−Xyz−Gly
The protein sequence following -Pro on the C-terminal side is revealed in various compositions in columns 1, 4, 6 and 11 below. Examples 1, 4, 6 and 11 show that the tetrapeptide sequence can be present at any position in the protein. However, when the product is a heterologous protein, the tetrapeptide sequence Pro-Xyz-Gly-Pro is always present on the N-terminal side of the heterologous protein.

By this method, not only Gly-Pro but also all dipeptide sequences linked at the amino terminus
Yzx-Pro can be cleaved from proteins.
This is because post-proline dipeptidyl aminopeptidases are not specific for the amino acids located before proline.

This method can also be used on the principle of so-called "direct synthesis" to convert the first fusion protein into the desired heterologous protein. A proline codon must be inserted in the heterologous gene between the methionine codon required as a starter and the nucleotide sequence encoding the protein. The first product is a protein extending towards the Met-Pro at the amino terminus, from which the Met-Pro sequence can be easily cleaved using post-proline dipeptidyl aminopeptidase. Again, this method can only be used if the peptide sequence that follows C-terminally with respect to proline does not begin with proline or aminoacylproline.

Xyz or Yzx are all naturally occurring amino acids contained in the genetic code.
Uvw is all naturally occurring amino acids except proline.

Amino terminal residue Gly-Pro, Met-Pro or
Yzx-Pro, N-terminal tripeptide sequence Yzx-
Cleavage from a fusion protein with Pro-Uvw using post-proline dipeptidyl aminopeptidase is carried out as follows: The fusion protein is cleaved in a suitable buffer, e.g. Tris buffer or phosphate buffer, depending on its solubility properties. It is dissolved in a liquid (PH range PH 7-8 being particularly advantageous) and isothermated with PPDA at 30-40 DEG C., in particular about 37 DEG C. The reaction time is largely determined by the fusion protein, and 3 to 5 hours is often sufficient.

In the present invention, other amino acids Zxy (with the exception of proline) are replaced by the tetrapeptide sequence Pro-Xyz-Gly-
Pro (Xyz may represent each arbitrary amino acid)
A heterologous protein having proline as the second N-terminal amino acid by introducing between the desired heterologous protein and the advantageous post-proline dipeptidyl after first collagenase cleavage of the Xyz-Gly-bond from the fusion protein. Aminopeptidase is used to cleave the Pro-Zxy-bond and then the amino acid Zxy
It is also possible to produce it by removing it with leucine aminopeptidase.

Having proline as the second N-terminal amino acid means that the heterologous protein has proline as the second amino acid at the N-terminus: i.e. For example, if the desired heterologous protein contains an additional proline as a second amino acid at the N-terminus, as in the case of human growth hormone or bovine prolactin, PPDA can degrade the protein into short proline-containing dipeptides. will continue.

By the way, using the above method, a tetrapeptide sequence in which Xyz may represent each arbitrary amino acid can be obtained.
Fusing a heterologous protein with proline as the second N-terminal amino acid when introducing another amino acid Zxy, which may be any amino acid other than proline, between Pro-Xyz-Gly-Pro and the desired heterologous protein. It can be obtained from protein. After collagenase cleavage of the Xyz-Gly bond of the pentapeptide sequence Pro-Xyz-Gly-Pro-Zxy, the dipeptide Gly-Pro can be cleaved using the enzyme PPDA, and proline-containing heterologous proteins are not affected. .
Thereafter, the protecting group Zxy is enzymatically cleaved using leucine aminopeptidase (LAP, EC3.4.11.1.). Using this enzyme, “direct synthesis”
The N-terminal methionine can also be cleaved from a fusion protein containing the sequence Met-Uvw-Pro, where Uvw can be any amino acid except proline. Conventionally, only the bromcyan method of SB Needleman (cited above), which involves side reactions, has been used for this purpose.

The advantages of high enzymatic activity and easy purification of cleavage products of PPDA can be exploited by introducing other amino acids Zxy (other than proline), such as leucine or glycine, between the collagenase specific sequence and the heterologous protein sequence. , can be used to obtain a desired heterologous protein having the N-terminal sequence Uvw-Pro, where Uvw represents the one described above.

In this case, a collagenase-specific nucleic acid sequence;
An additional codon for the amino acid Zxy must be introduced between the nucleic acid sequence encoding the heterologous protein.

Amino acids Zxy are all naturally occurring amino acids other than proline that are included in the genetic code.

When the protein of interest has an N-terminal proline residue, the method of the present invention can be applied in a similar manner. In the case of the fusion protein, this N-terminal proline residue already represents the proline residue following Gly on the carboxyl-terminal side. Collagenase specificity sequence Por
−Xyz−Gly−Pro to Pro−Xyz by collagenase and aminoacylproline aminopeptidase
and in the case of cleavage of Gly and “direct synthesis”
The methionine residue is removed from the fusion protein starting at the Met-ProN-terminus using aminoacylproline aminopeptidase.

The heterologous protein extending towards the amino acid at the N-terminus is prepared in a suitable buffer such as Tris buffer or phosphate buffer (PH 8-10, especially 8.6).
For activation of LAP, 1-5 mmol/MnCl ₂ is generally added to dissolve and after addition of LAP.
Maintain constant temperature at 35-40℃, especially at 40℃. The reaction time depends significantly on the N-terminal amino acid to be cleaved;
It may be for several minutes to several hours.

The desired heterologous protein, which is present in free form, is isolated in pure form by conventional protein purification techniques. The method used in this case is to use a foreign protein (e.g. proinsulin, insulin A chain or insulin B chain,
ACTH, STH) and the characteristics of the compound to be separated.

It is clear from the Examples that the enzyme post-proline dipeptidyl aminopeptidase accepts not only average peptides but also high-molecular proteins as substrates.

Tetrapeptide sequences Pro of Examples 1, 4, 6 and 11
−Xyz−Gly−Pro is: Example 1 140 mg (50 μmol) of Z-Gly-Pro-Leu-Gly-Pro-insulin A chain to avoid hydrolysis at undesired locations due to collagenase contamination with 0.1 mol of calcium acetate/and other proteases. Diisopropylfluorophosphate
Dissolve in 10 ml of 0.5 mol/- Tris buffer (PH 7.2) containing 0.1 mmol/- and add thereto 10 mg of collagenase. After 60 minutes at 28°C, the reaction is stopped by the addition of ethanol. After separation of the precipitated enzyme, the alcohol is almost evaporated in vacuo and the solution is desalted by chromatography on Sephadex G-15 using 0.01 mol/- ammonium bicarbonate as eluent. The eluate is concentrated to a small volume in vacuo and lyophilized. The yield of Gly-Pro-insulin A chain is
It is 110mg. N- by end group measurement (DNP method)
Glycine can be detected as an amino acid. The product obtained is used directly in further reactions.

Example 2 Cleavage of both first N-terminal amino acids separately 50 mg of Gly-Pro-insulin A chain was dissolved in MnCl ₂ 0.5
Dissolve in 1 ml of 0.1 mol/- Tris buffer (PH 8.5) containing mmol/-. Add 2 μg of aminopeptidase-P and incubate at 37° C. for 1 hour. 20 μg of proline-aminopeptidase is inactivated by brief heating to 60° C. and added without further purification. After 24 hours at 37° C., the high molecular weight proteins are precipitated with alcohol and separated by filtration. Concentrate the eluate in vacuo and
0.01 mol/ammonium bicarbonate buffer (PH
9) and desalted by chromatography using Sephadex G-25. Pour the insulin A chain fraction directly onto an AE-cellulose column and add an ammonium bicarbonate gradient (0.01~
Perform chromatography treatment at 0.1 mol/pH 9). Concentration and lyophilization of the insulin A chain fraction yields 40 mg of insulin A chain.

Amino acid analysis: Calculated value Asp2.00 Thr1.00 Ser2.00 Glu4.00 Gly1.00 Cys4.00 Val1.00 Ile2.00 Leu2.00 Tyr2.00 Actual value Asp1.98 Thr0.92 Ser1.64 Glu3.79 Gly1 .02 Cys cannot be measured Val0.98 Ile1.99 Leu2.04 Thr1.86 Example 3 Cleavage of both first N-terminal amino acids together Gly-Pro-insulin A chain 50 as in Example 2
mg is dissolved in 1 ml of Tris buffer. Then 2 μg of aminopeptidase-P and 20 μg of proline aminopeptidase are added. Reaction time at 37℃
After 18 hours it is precipitated with alcohol and worked up as in Example 2.

Yield: 30 mg of insulin A chain Example 4 200 mg of Z-Gly-Pro-Leu-Gly-Pro-insulin B chain is reacted in the same manner as in Example 1. Gly-Pro
- Yield of insulin B chain is 150 mg, which is further processed without purification.

Example 5 100 mg of Gly-Pro-insulin B chain prepared according to Example 4 was dissolved in 5 ml of Tris buffer, and kept at constant temperature with aminopeptidase-P and proline aminopeptidase back and forth in the same manner as in Example 2. Desalt and purify by chromatography.

Yield: Insulin B chain 70mg Amino acid analysis: Calculated values: Asp1.00 Thr1.00 Ser1.00 Glu3.00 Gly3.00 Pro1.00 Cys2.00 Phe3.00 Val3.00 Leu4.00 Ala2.00 Tyr2.00 His2. 00 Lys1.00 Arg1.00 Actual value: Asp0.95 Thr0.92 Ser0.83 Glu3.12 Gly3.02 Pro0.98 Cys1.75 Phe2.98 Val3.10 Leu3.96 Ala2.04 Tyr1.89 His1.94 Lys1 .06 Arg0.97 Example 6 Z−Gly−Pro−Gly−Gly−Pro−Ala−Met−
15 mg of Glu-His-Phe-Arg-Trp-Gly are reacted in the same manner as in Example 1.

Yield: Gly−Pro−Ala−Met−Glu−His−Phe
-Arg-Trp-Gly10mg Example 7 Gly-Pro-Ala-Met- produced according to Example 6
10 mg of Glu-His-Phe-Arg-Trp-Gly are reacted as in Example 3 together with aminopeptidase-P and proline aminopeptidase. For purification, the enzyme is precipitated with ethanol, concentrated to a small volume and chromatographed over carboxymethyl cellulose using an ammonium acetate gradient (0.01-0.2 mol/).

Yield: Ala−Met−Glu−His−Phe−Arg−Trp
−Gly7mg Amino acid analysis: Calculated value: Glu1.00 Gly1.00 Phe1.00 Ala1.00 Met1.00 His1.00 Arg1.00 Trp1.00 Actual value: Glu1.03 Gly1.01 Phe0.98 Ala1.02 Met0.81 His0.93 Arg0・97 Trp Unmeasurable example 8 50 mg of Gly-Pro-Ala was added to post-proline dipeptidyl aminopeptidase (hereinafter referred to as PPDA)
Tris buffer (20 mmol/,
PH7.8) Maintain constant temperature at 37°C for 3 hours in 2 ml. Add 1 ml of ethanol to the cleavage batch to remove the enzyme and separate the precipitated enzyme. After concentrating the liquid
The mixture is separated by taking up in NH ₄ HCO ₃ buffer (0.01 mol/, PH 7.4) and chromatographing through a cellulose column. After the corresponding fractions have been combined, they are concentrated in vacuo and freeze-dried twice.

Yield: Alanine 13 mg (70%) Example 9 Gly-Pro-Ser-Tyr-βNA (βNA: β-naphthylamide) is kept at constant temperature in the same manner as in Example 8 with 700 μg of PPDA. To separate the mixture, the Ser-Tyr-βNA formed is extracted with acetate and, after concentration, precipitated twice from methanol/ethanol.

Yield: Ser-Tyr-βNA 43 mg (74%) Melting point: approx. 205℃ (decomposed) Example 10 Gly-Pro-insulin A chain-tetra-S-
Dissolve 50 mg of sulfonate (bovine) in 2 ml of Tris buffer (0.1 mol/, pH 8.0) and add 100μ of PPDA.
Add g. After 4 hours at 37℃, cephadex
0.01 mol/- as eluent via G50
Perform chromatography using NH ₄ HCO ₃ (PH7.5). After the corresponding fractions have been combined, they are concentrated to a small volume and freeze-dried twice.

Yield: 43 mg insulin A chain This product, unlike the starting material, can be degraded with leucine aminopeptidase.

Amino acid analysis: Calculated value: Asp2.00 Ser2.00 Glu4.00 Gly1.00 Cys4.00 Val2.00 Ala1.00 Actual value: Asp1.97 Ser1.70 Glu4.02 Gly1.05 Cys cannot be measured Val1.99
Ala0.97 Calculated value Ile1.00 Leu2.00 Tyr2.00 Pro0.00 Actual value 1.04 2.08 1.78 0.04 Example 11 Prolactin-hexa-S-sulfonate 20 mg
was dissolved in 5 ml of Tris buffer (0.5 mol/, PH 7.2) and calcium acetate 0.1 mol/, diisopropylfluorophosphate 0.1 mmol/
and 5 mg of collagenase. After 30 minutes at 30℃,
The reaction is stopped by adding ethanol. After concentration in vacuo, Tris buffer (0.02 mol/, PH
7.8) Chromatograph through Sephadex column G75 using the same buffer as eluent. The combined fractions are desalted by dialysis against demineralized water and, after lyophilization, taken up in 4 ml of Tris buffer (0.1 mol/pH 7.9) and incubated with 5 μg of PPDA at 37° C. for 2 hours. It is chromatographed again via Sephadex G75, the corresponding fractions are combined, dialyzed and freeze-dried.

Yield: 10 mg of desoctapeptide-prolactin-penta-S-sulfonate Unlike the starting material, Gly can be detected as the N-terminal amino acid. Furthermore, the final product, unlike the intermediate products, can be decomposed by LAP.

Example 12 150 mg of Met-Pro-Ser-Tyr-βNA are reacted and worked up as in Example 2 with 1 mg of post-proline dipeptidyl aminopeptidase.

Yield: Ser-Tyr-βNA 80 mg (70%) Melting point: approx. 208°C (decomposed) Example 13 500 mg of Met-Gly-Pro-amide was mixed with 1 mg of active leucine aminopeptidase in Tris buffer (30%).
mmol/- Tris HCl, 2 mmol/-
MnCl ₂ , PH8.6) in 20 ml and kept constant at 40°C for 30 minutes. The Gly-Pro-amide is shaken out with acetate in the cold and, after concentration in vacuo, reprecipitated twice from ethanol/petroether.

Yield: 242 mg〓72% Melting point: 209-210℃ (decomposed) [α] _D = 194.4 (c = 1, H ₂ O) Example 14 100 mg of bovine growth hormone was added to Tris buffer (30 mmol/-Tris.HCl, 2 mmol/-
Incubate with 25 μg of activated leucine aminopeptidase in 10 ml (MnCl ₂ , PH 8.6) at 40° C. for 30 minutes. The proteins were then precipitated with ethanol,
Bicarbonate buffer (10 mmol/ _{-NH4HCO3 ,}
PH7.5) and the mixture is separated at 4° C. through a Sephadex G50 column (100 cm×1 cm (φ)) with the same bicarbonate buffer. Fractions containing desalanyl ¹ -growth hormone are combined, concentrated and lyophilized. After dansylation and acid hydrolysis, phenylalanine is identified as the terminal amino acid by thin layer chromatography.

Yield: 84mg〓84% Example 15 Gly−Pro−Lew−Gly−Pro−amide 100mg
The temperature is maintained at 37°C for 3 hours in 1 mg of PPDA and 5 ml of Tris buffer (20 mmol/-Tris.HCl, pH 7.3). The Lew-Gly-Pro-amide formed is shaken out with acetate in the cold. After concentration, add Tris buffer (30 mmol/-Tris・HCl,
0.1 mg of activated _leucine aminopeptidase was added thereto, and the mixture was incubated at 40° C. for 30 minutes. The final product is shaken out with acetate in the cold and, after concentration in vacuo, is precipitated twice from ethanol/petroether.

Yield: 18.3mg〓47% Melting point: 209-210℃ (decomposition) [α] _D = 194.4 (c = 1, H ₂ O)

Claims (1)

[Claims] 1. In a protein containing a tetrapeptide sequence Pro-Xyz-Gly-Pro in any position, in which Xyz may represent any amino acid, an Xyz-Gly bond in the tetrapeptide sequence is selected by collagenase. 1. A method for producing a protein, which comprises cleaving the protein directly, followed by removing glycine residues using aminoacylproline-aminopeptidase and removing proline residues using proline aminopeptidase. 2. The method according to claim 1, wherein the cleavage of glycine residues with aminoacylproline-aminopeptidase and the cleavage of proline residues with proline aminopeptidase are carried out in one step. 3. In a protein containing the tetrapeptide sequence Pro-Xyz-Gly-Pro in any position, where Xyz may represent any amino acid, the Xyz-Gly bond in the tetrapeptide sequence is selectively cleaved with collagenase, and then A method for producing a protein, which comprises cleaving a Gly-Pro residue using post-proline dipeptidyl aminopeptidase. 4. In a method for producing a heterologous protein containing proline as the second N-terminal amino acid, Xyz
In a protein containing the tetrapeptide sequence Pro-Xyz-Gly-Pro, which may represent any amino acid, another amino acid Zxy, which may represent any amino acid other than proline, is added to the N-terminal tetrapeptide sequence Pro- After introducing Xyz-Gly-Pro and a desired heterologous protein and cleaving the Xyz-Gly bond with collagenase, the Pro-Zxy bond is cleaved with post-proline dipeptidyl aminopeptidase, and then the amino acid Zxy is replaced with leucine aminopeptidase. A method for producing a protein characterized by being cleaved with peptidase.