JPH0320237B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing insulin having the same primary structure as human insulin (hereinafter referred to as "human insulin"). Concerning a method for semi-synthesizing.

Insulin is used as a treatment for diabetes in cattle,
It is a protein hormone that is industrially produced and used from pigs, whales, bonito, etc. Insulin of humans and most higher animals has a peptide chain consisting of 21 amino acids called the A chain and a peptide chain consisting of 30 amino acids called the B chain, and there is a peptide chain between the A chain and the B chain. It is bonded by two disulfide bonds (-S-S-).
Furthermore, there is one disulfide bond in the A chain. The amino acids constituting each peptide chain and their sequences differ depending on the species of animal, but human insulin has extremely similar structures to those of pigs, sperm whales, fin whales, and rabbits. The drawing shows 1 of human insulin.
The following structure shows that the amino acid residue at the C-terminus (B ₃₀ ) of the B chain is threonine (Thr) in humans, whereas it is alanine (Ala) in pigs, sperm whales, and fin whales, and serine in rabbits. The only difference is that it is (Ser).

Therefore, if the amino acid residue at the C-terminus of the B chain of insulin from pigs, sperm whales, fin whales, rabbits, etc. (hereinafter collectively referred to as "porcine insulin") is replaced with Thr, a human version with the same primary structure as human insulin can be obtained. Insulin can be synthesized. Such human insulin has less antigenicity than insulin preparations conventionally obtained from xenogeneic animals, making it possible to produce highly safe insulin preparations.

In the past, attempts have been made to produce human insulin from porcine insulin, focusing on the above facts, but most of these attempts involve arginine-glycine (Arg-glycine), which is cleaved by trypsin, in the B chain of insulin. Gly) bond (B ₂₂ −
Desoctapeptide _{-B23 - B30} -insulin (referred to as "DOI")
was produced, and this was used as a raw material to condense it with the octapeptide (B ₂₃ -B ₃₀ ) of human insulin to produce human insulin. These conventionally proposed production methods are not necessarily satisfactory, as they are limited by the technological level of each era and pose problems in the protection techniques for amino groups and carboxyl groups. In particular, since it passes through DOI as an intermediate product, it is necessary to synthesize an octapeptide to be condensed with it. However, efficient synthesis of this octapeptide was accompanied by technical difficulties. Additionally, there was concern that residues of trypsin used in the DOI production stage would have an adverse effect on the octapeptide and condensation stage.

Therefore, the purpose of the present invention is to use Pro (proline)-Lys (lysine) of the B chain as an enzyme to replace trypsin.
(B ₂₈ −B ₂₉ ) To provide an efficient method for producing human insulin by cleaving a peptide bond using an enzyme that specifically acts on the bond and then generating the peptide bond using the same enzyme. It is in. Since the present invention uses the same enzyme for cleavage and generation of peptide bonds, there is no possibility of adverse effects of foreign enzymes or catalysts. Furthermore, since the peptide used in the production step is a short dipeptide (Lys-Thr), it is easy to synthesize, making the production method of the present invention extremely practical.

The method for producing human insulin of the present invention involves converting porcine insulin into Pro-
a step of hydrolyzing in the presence of an enzyme that specifically acts on Lys bonds to produce desdipeptide-B ₂₉ , B ₃₀ -insulin (hereinafter abbreviated as “DDI”);
_X2 -desdipeptide- _B29 , _B30 -insulin (hereinafter abbreviated as " _X2 -DDI") and N〓-X-Lys-
The method is characterized in that it includes a step of condensing Thr (X represents an amino protecting group that can be decomposed by proton solvolysis or β-elimination) in the presence of the same enzyme as in the above step.

“Porcine insulin B chain” used in the present invention
"Enzyme that specifically acts on Pro-Lys bond" (hereinafter referred to as "Enzyme that specifically acts on Pro-Lys bond"
Examples of the enzymes (abbreviated as "enzymes of the present invention") include the following.

(1) R, Walter isolated from lamb kidney and Biochimica et Biophysica
Acta, 422 (1976) 138-158.

(2) Peptidases (Post-proline
cleaving enzyme) with the following enzymatic properties:

Action and substrate specificity It acts specifically on and decomposes specific proline-related peptide bonds.

a What is decomposed (↓ indicates the bond to be decomposed) Z−Gly−Pro−↓2−NNap Z−Ala−↓Pro−2−NNap Z−Gly−Pro−↓ONp Z−Gly−Pro−↓ MCA Z−Gly−Pro−↓Ala Z−Gly−Pro−↓Leu−Gly Z−Gly−Pro−↓Leu Z−Gly−Pro−↓Leu−Ala Z−Gly−Pro−↓Phe Z−Gly−Pro −↓Leu−D−Ala Pht−Gly−Pro−↓Leu−Gly Z−Gly−Pro−↓Leu−Gly−Gly For−Gly−Pro−↓Leu−Gly Z−Gly−Pro−↓Leu−Gly− Ala Thr−Lys−Pro−↓Arg Z−Gly−Pro−↓Leu−Gly−Pro Cys−Tyr−Phe−Gln−Asn−Cys−Pro
−↓Arg−Gly−NH ₂ Cys−Tyr−Ile−Gln−Asn−Cys−Pro−↓
Leu−Gly−NH ₂ b Not decomposed Gly−Pro−2−NNap Z−Pro−2−
NNap Pro−2−NNap Gly−Pro−Leu
−Gly Z−Gly−Pro−D−Ala Z−Ala
−Gly−Pro Z−Pro−Leu Gly−Pro
Gly−Pro−Ala Leu−Gly Gly−Gly−
Gly Z-Gly-Phe In the above formula, Z is carbobezonexy group, 2-NNap is β-naphthylamide, ONp is p-
Nitrophenyl ester, Pht represents a phthalyl group, and For represents a formyl group.

Optimal action PH 7.0 Stable PH range 5.0-9.0 Optimum action temperature 40°C Thermostability 42°C or less Inhibitory action Not inhibited by pancreatic and limabean trypsin inhibitors, ovomucoid, pepstatin, etc.

Molecular weight: approximately 78,000 Isoelectric point: PH9.1 Examples of bacteria producing the above enzyme include Flavobacterium meningosepticum.
IFO12535, Flavobacterium TY−78−74
(Flavobacterium TY-78-74) Microtechnical Research Institute No. 4849 is available.

Flavobacterium TY-78-74 has the mycological properties shown below.

Morphology 1 Cell shape: Bacillus 2 Motility: None 3 Gram staining: - Growth 1 Broth agar plate culture: + 2 Broth agar slant culture: + 3 Broth liquid culture: + Physiological properties 1 Nitrate reduction: + 2 Indole Production: + 3 Hydrogen sulfide production: - 4 Starch decomposition: - 5 Inorganic nitrogen source utilization: - 6 Pigment production: Yellow 7 Urease: + 8 Oxidase: + 9 Catalase: + 10 Growth temperature range: 20~ 40℃ 11 Oxygen: Aerobic 12 OF test: 0 or + 13 Gas evolution a L-arabinose: - b D-xylose: - c D-glucose: + d Sucrose: - e Lactose: + f D-mannite :+ g Starch:- (3) William G.Lewis
isolated from Basidiomycete Armillaria mellea and called it Armillaria mellea protease, Biochimica et Biophysica Acta, 522
(1978) 551-560.

(4) M. Wingard et al. J.
Myxobacter AL-1 protease reported in Bacteriol. 112 (1972) 940.

The reaction of cleavage of Pro-Lys of the B chain of porcine insulin in the presence of the enzyme of the present invention is carried out in an aqueous medium for 4 to 48 hours as usual at a temperature of 30 to 40°C.
C. and pH of 7 to 9 are appropriate. More preferable conditions for each enzyme used are 30 to 40°C and pH 7.5 for the peptidase isolated from lamb kidney mentioned in (1) above.
~8.0, and in the case of enzyme (2), 30~40℃,
PH7.0-8.0. In the case of (3) Armillaria melea protease, the temperature is 30-40℃, pH7.0-
8.0 is preferred. Through this process, the dipeptide Lys-Ala or Lys at the C-terminus of the B chain of porcine insulin
-Ser is cleaved from the main chain and DDI is generated. The reaction product thus obtained was
50 (trade name, manufactured by Pharmacia), to separate Lys-Ala or Lys-Ser, and collect the DDI fraction.

Next, the amino groups at the N-terminals of both A and B chains of DDI were converted into tertiary butyloxycarbonyl residue (BOC)-, tertiary amyloxycarbonyl residue (AOC)-, methylsulfonyl Ethyloxycarbonyl residue (MSC) - an amino protecting group (X) that can be decomposed by proton solvolysis or β-elimination
protected by X ₂ −DDI.

Next, a condensation reaction between X ₂ -DDI and N–-X-Lys-Thr is caused in the presence of the same enzyme as in the cleavage step. In this case, reaction conditions must be selected so that the enzyme exhibits transpeptidase activity and the equilibrium is tilted toward synthesis. The conditions are that N〓-X-Lys-Thr be present in an excess amount, that is, approximately 10 times the molar amount or more, relative to X ₂ -DDI, that the concentration of the reactant be as high as possible, and that the reaction product be immediately It is important to exclude it from the reaction system. Specific examples include adding an organic solvent such as dimethylformamide (DMF), dimethyl sulfoxide (DMSO), methanol, etc. to water as the reaction medium. The proportion of organic solvent addition is 40-60%
is preferred. Further, the temperature is preferably 30 to 40°C and the pH is preferably 6 to 7.

Hereinafter, a more detailed explanation will be given based on examples.

Example 1 (a) 570 mg (100 μmole) of pork insulin and Flavobacterium meningosepticum
Peptidase mentioned above (2) obtained from IFO12535
0.6mg/10.5U) in 0.05M phosphate buffer (PH
8.0) Dissolved in 30 ml and reacted at 30°C for 20 hours.
Then, after cooling on ice, adjust the pH to 2 with 3NHCl, and apply a Sephadex G-50 column (column size 4.0 x 140 cm,
Elute with 0.01M HCl, flow rate 87.2ml/hr), separate the proline peptidase used and Lys-Ala produced, collect 490ml of insulin residual fraction eluted from 1040ml to 1530ml,
Freeze-drying yielded 495 mg (90 μmole, yield 90%).

(b) 495 mg of this insulin residual fraction was mixed with 65 ml of dimethylformamide, 108 mg of triethylamine,
Boc−N ₃ (t-Butyloxycarbonyl-azide)4.3
Dissolve in g and stir at 40℃ for 1 hour to react.
By precipitating with ether (Boc) ₂ -insulin residual fraction conjugate 510 mg (90 μmole,
A yield of 100% was obtained.

(c) Synthesis reaction () 510 mg (90 μmole) of the above conjugate and N〓−Boc
-Lys-Thr315mg (900μmole) 0.05M
It was dissolved in NH ₄ HCO ₃ and freeze-dried.

() After dissolving this freeze-dried product in 1.7 ml of DMF (dimethylformamide), 1.7 ml of 0.25M Tris-HCl buffer was added to adjust the pH to 6.5.

() 7 mg of the same peptidase used in (a)
(122.5 U) was added at 3.5 mg each at the start of the reaction and at 8 hours, and the reaction was carried out at 37°C for 24 hours.

() Add 0.7ml of 0.4M HCl to make it acidic (PH3.5) to stop the reaction, and add Cephadex LH-
20 column (column size 2.5 x 96.0 cm), and perform chromatographic fractionation by elution with a DMF-0.5M acetic acid (1:1) solution (flow rate 23.4 ml/hr). Through this chromatographic fractionation, 280 mg of residual N-Boc-Lys-Thr that did not participate in the reaction was recovered (recovery rate 89%).

() generated (Boc) ₃ -insulin with TFA
(trifluoroacetic acid) 5ml and anisole 1ml
The Boc group was removed by reacting with a mixture consisting of the following at 0°C for 60 minutes.

() DEAE− produced insulin was buffered with 0.02M ammonium acetate buffer PH7.6.
Sephadex A-25 column (column size
2.0 x 42 cm), elute and fractionate by linearly increasing ammonium acetate buffer.

Human insulin yield: 260 mg (yield: 50%) Insulin residual fraction yield: 190 mg (yield:
(37%) (d) Comparative identification of the manufactured human insulin and natural human insulin was performed using the following method.

() Amino acid composition analysis () HPLC (high performance liquid chromatography) () Polyacrylamide gel electrophoresis () Biological test: hypoglycemic effect, etc. All results showed that there was no significant difference between the two. .

Example 2 (a) 60 mg of sperm whale insulin and 0.4 mg of the peptidase obtained from the above (1) from lamb kidney.
(6.0U) in 0.1M sodium acetate buffer (PH7.5)
Dissolve in 6 ml and react at 40℃ for 35 hours, cool on ice, adjust pH to 2.6 with glacial acetic acid, and add Cephadex G.
-50 column (column size 2.5 x 96 cm, flow rate
23.4ml/hr, eluted with 0.5M acetic acid),
The insulin residual fraction (140 ml eluted from 280 ml to 420 ml) was collected by molecular sieve chromatography and lyophilized to obtain 44 mg (yield 80%).

(b) A (Boc) ₂ -insulin residual fraction conjugate was synthesized using Boc-N ₃ by a conventional method.

Yield 45 mg (yield 100%) (c) 45 mg of the above conjugate and 22 mg of N〓−Boc−Lys−Thr
was dissolved in 0.05M NH ₄ HCO ₃ and lyophilized.
After dissolving in 0.2ml of DMSO (dimethyl sulfoxide)
Add 0.2ml of 0.25M Tris-HCl buffer (PH6.5
4.6 mg of the same peptidase used in (a) was added to this solution at the start of the reaction and 2.3 mg at 6 hours, and the reaction was allowed to proceed at 40°C for 24 hours.

After adding 0.1 ml of 0.4M HCl to the reaction solution to make it acidic, it was added to a Sephadex LH-20 column (1.6 x 96.0
Perform chromatographic fractionation using a DMSO-0.5M acetic acid (1:1) solution. Obtained (Boc) ₃ −
After treating insulin with TFA/anisole, a DEAE-cellulose column (column size:
1.6 x 17 cm) and elution fractionation was carried out by linearly increasing the NaCl concentration (0M→0.6M) to obtain human insulin.

Human insulin yield: 19 mg (yield: 41%) Insulin residual fraction yield: 20 mg (yield: 45%)
%) (d) Comparative identification of the manufactured human insulin and natural human insulin was performed using the following method.

() Amino acid composition analysis () HPLC (high performance liquid chromatography) () Polyacrylamide gel electrophoresis () Biological test: hypoglycemic effect, etc. All results showed that there was no significant difference between the two. .

[Brief explanation of the drawing]

The drawing represents the primary structure of human insulin.

Claims (1)

[Claims] 1. A peptidase isolated from lamb kidney that specifically acts on the Pro-Lys bond of the B chain of porcine insulin, Flavobacterium meningosepticum IFO 12535 or Flavobacterium TY.
A peptidase produced by -78-74 with the following enzymatic properties: A. Action and substrate specificity: Acts specifically on a specific proline-related peptide bond. a What is decomposed (↓ indicates the bond to be decomposed) Z−Gly−Pro−↓−2NNap Z−Ala−Pro−↓2−NNap Z−Gly−Pro−↓ONp Z−Gly−Pro−↓MCA Z−Gly−Pro−↓Ala Z−Gly−Pro−↓Leu−Gly Z−Gly−Pro−↓Leu Z−Gly−Pro−↓Leu−Ala Z−Gly−Pro−↓Phe Z−Gly−Pro− ↓Leu−D−Ala Pht−Gly−Pro−↓Leu−Gly Z−Gly−Pro−↓Leu−Gly−Gly For−Gly−Pro−↓Leu−Gly Z−Gly−Pro−↓Leu−Gly−Ala Thr−Lys−Pro−↓Arg Z−Gly−Pro−↓Leu−Gly−Pro Cys−Tyr−Phe−Gln−Asn−Cys−Pro
−↓Arg−Gly−NH ₂ Cys−Tyr−Ile−Gln−Asn−Cys−Pro−↓
Leu−Gly−NH ₂ b Not decomposed Gly−Pro−2−NNap Z−Pro−2−
NNap Pro−2−NNap Gly−Pro−Leu
−Gly Z−Gly−Pro−D−Ala Z−Ala
−Gly−Pro Z−Pro−Leu Gly−Pro
Gly−Pro−Ala Leu−Gly Gly−Gly−
Gly Z-Gly-Phe In the above formula, Z is a carbobenzoxy group, 2-
NNap is β-naphthylamide group, ONp is p-nitrophenyl ester residue, Pht
represents a phthalyl acid residue, and For represents a formyl group. B Optimum action PH 7.0 Stable PH range 5.0-9.0 C Optimum action temperature 40°C D Thermostability 42°C or less E Inhibitory action Not inhibited by pancreatic and limavian trypsin inhibitors, ovomucoid, and pepstatin. F Molecular weight: approximately 78,000 G Isoelectric point: PH9.1 The insulin is hydrolyzed in the presence of peptidase produced by Basidiomycete armillaria melea or protease produced by Myxobacter AL-1, and desdipeptide-B ₂₉ , B ₃₀ - _a step _of producing _insulin ; A method for producing human insulin, comprising a step of condensing it in the presence of a human insulin.