CN1099416A - Process for gene engineering production of IL 28-GLP-I - Google Patents

Abstract

This invention adopts high density fermentation of Il328-GLP-I(7-36) gene engineering strain post-treatment and modification, as well as concentration, purification of expression product of the engineering strain. The determination shows that by utilization of this product produced by this tochnology, the secretion of insulin from animal pancreas can be significantly increased. The scale production of GLP-I drug by gene engineering may be realized.

Description

Process for gene engineering production of IL 28-GLP-I

L-iLeu of the present invention ₂₈The process for gene engineering production of-GLUCAGON LIKE PEPTIDE-I (being polypeptide Ile28-GLP-I (7-36)) relates to polypeptide drugs and genetically engineered.

Diabetes are one of common diseases of people, and the patient is divided into two types, insulin-dependent diabetes mellitus (IDDM) patient, pancreatic beta cell lost the excreting insulin worker can, thereby need insulin injection to treat; And the type II diabetes patient, its pancreatic beta cell is not lost the function of excreting insulin, does not but know what reason, still can not if can make the β cell with the normal amount excreting insulin with the normal amount excreting insulin, and then type II diabetes just can obtain medical treatment.But up to now, also there is not suitable medicine can promote β emiocytosis Regular Insulin single-mindedly.Therefore, still there is not effectively to treat the medicine of type II diabetes.At present, whole world diabetic subject is about more than 100,000,000, and the 2-3 that the U.S., Canada, Australia, European various countries type II diabetes patient are the insulin-dependent diabetes mellitus (IDDM) patient doubly; The diabetic subject of Japan, the II type is 10 times of I type:

The situation of China, according to the academic meeting material of the 5th diabetes of in November, 1992 China medical association, the type II diabetes patient accounts for more than 90%, is mainly the elderly, and insulin-dependent diabetes mellitus (IDDM) patient is mainly children and teenager, accounts for diabetic subject's 5% altogether.From diabetic subject's statistics of Japan and China, Asia diabetic subject's ratio is based on the type II diabetes patient, and Europe, North America, Australian all states II type are 3: 1 with the ratio of I type.Therefore for providing effective medicine, type II diabetes has crucial meaning.

Treat several schemes of type II diabetes (Japan) at present, regimen accounts for 35%, and insulin injection mainly slowly to continue the delivery mode administration, accounts for 25%, oral hypoglycemic 40%.

Glucose in the food, amino acid, lipid acid etc., wherein glucose can be urged the pancreatic beta cell excreting insulin, and these diet group compositions can also promote intestinal secretion inertin to promote secretion of insulin.Early stage result of study proves, the oral back of identical glucose promotes the secretion of insulin effect than strong many of intravenous promotion secretion of insulin effect, estimate that food promotes insulin secretion on, the hormone role of intestinal secretion may account for over half.

In the polypeptide hormone of intestinal secretion except that Regular Insulin, also has hyperglycemic-glycogenolytic factor (Glucagon), Somat (Somatotin), Proglucagon precursor (Preproglucagon) etc., the processing through proteolytic ferment in small intestine and pancreas of Proglucagon precursor is modified, form polypeptide: GLP-I (7-37)-OH, GLP-I (7-36)-NH ₂, GLP-II etc. wherein have only GLP-I (Glugon Like peptide I) in the presence of glucose, 5 * 10 ^-11The M lower concentration can promote the pancreas excreting insulin effectively.

Owing to contain the characteristic of GLP-I gene class polypeptide institute tool, following advantage can be arranged as treatment type II diabetes medicine:

1) under the physiological concentration condition, can promote the pancreas excreting insulin single-mindedly,

2) promote how much deciding with the concentration of glucose in the blood of pancreas excreting insulin, islet secretion is many when blood sugar concentration is high, when blood sugar concentration is hanged down, insulin secretion is few, therefore unlike insulin injection, often cause hypoglycemia, even suffer a shock, take fool proof.

3) be that material long-term the taking unlike present type II diabetes medicine Suifonylurea that exists naturally in the human body can be destroyed the pancreas cell.

4) can also suppress glycogenolysis and become glucose, thereby reduce the glucose content in the blood indirectly.

5) can utilize genetic engineering technique, carry out mass production.

1992.6.6, and 1992.6.2, disclosing the patent No. respectively is U.S.5,120,712 and U.S.5, two parts of United States Patent (USP)s of 118,666, summary of the invention are crude substance GLP-I, i.e. (7-37) OH and (7-36)-NH ₂, its molecular structure is:

GLP-Ⅰ（7-37）OH

His-Ala-Glu-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly-OH

GLP-Ⅰ（7-36）-NH ₂

His-Ala-Glu-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Tle-Ala-Trp-Leu-Val-Lys-Gly-Arg-NH ₂

But because above-mentioned crude substance content in animal body is atomic, the extraction process complexity is difficult to mass production for practicality.So far also do not see that such polypeptide using gene engineering technique solves the report of the processing method of scale production.

The objective of the invention is to provide GLP-I class polypeptide Ile ₂₈The process for gene engineering production of-GLP-I (7-36).

The present invention is achieved in that

Production technique of the present invention is with fusion protein form expression, amino acid is engaged with fusion rotein carry out cracked technology again, and being characterized in adopting methionine(Met) is the juncture, carries out cracking with CNBr.Its flow process is:

Engineering bacterium fermentation

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Centrifugal collection thalline

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Ultrasonic disruption

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Centrifugation

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The supernatant precipitation

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Washing

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Cracking

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Concentrate

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Column chromatography

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High pressure liquid chromatography (HPLC)

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Product (lyophilized powder)

Wherein engineering bacteria is: Ile ₂₈-GLP-I (7-36) is made up of 30 amino-acid residues, and its sequence is:

His-Ala-Glu-GLY-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-ILe-Gly-Arg-X

Ester class, any amino acid, different lengths polypeptide, acid amides that X=free carboxyl group, free carboxyl group salt, medicine are accepted.

This genetic engineering bacterium has following feature: Ile ₂₈-GLP-I (7-36) polypeptide and A protein, beta-galactosidase enzymes, the plain CAT of chlorine enzyme, K ₈₈Fimbrial antigen albumen etc. are that the juncture forms fusion rotein with the methionine residues, and with the CHBr cracking, get product form is Ile to its fusion rotein under 70% formic acid or 8M urea/0.1 NHCl condition ₂₈Ester class, any amino acid, different lengths polypeptide, acid amides that-GLP-I (7-36), C termination have free carboxyl group, free carboxyl group salt, medicine to accept.

Use polypeptide gene engineering production technique of the present invention, not only in large-scale production, can make high yield, highly purified polypeptide Ile ₂₈-GLP-I (7-36) is with Glp-1(7-37) the same biologos with significantly short pancreas cell, and can be oral, its genetically engineered technology can be used for the production of other similar polypeptide equally.

The following drawings and embodiment will be further described the production technique of polypeptide PLG-I (7-37) genetic engineering bacterium of the present invention and expression product thereof:

Fig. 1 is the situation map that turns sour of the present invention

Fig. 2 is Ile ₂₈-GLP-I (7-36) Expression of Fusion Protein electrophorogram

Fig. 3 is Ile ₂₈-GLP-I (7-36) high pressure liquid chromatography (HPLC) analysis chart

The mensuration figure of Fig. 4 insulin standard product

Fig. 5 Ile ₂₈-GLP-I (7-36) is to rat pancreas promoting insulin secretion figure

Fig. 6 Ile ₂₈-GLP-I (7-36) and chemosynthesis GLP-I (7-37) are relatively to pancreas promoting insulin secretion figure

Fig. 7 Ile ₂₈-GLP-I (7-36) and the natural product GLP-of chemosynthesis I (7-37) are relatively to pancreas promoting insulin secretion figure

Embodiment 1,

1, ferment-seeded is cultivated:

Take out the PWSI of low temperature storage ₃₀/ JM103 engineering bacteria glycerine pipe, after the thawing, be inoculated in the containing in the penbritin 50 μ g/ml LB nutrient solutions of 250ml, inoculum size is the 1/50-1/100 volume, 35-37 ℃ vibrated 12-20 hour, was inoculated in the 10L fermentation culture as seed, and every ml fermented liquid adds ampicillin 50 μ g/ml, 35-37 ℃ of stirring, blowing air was cultivated 12-18 hour.

2, fermentation

Preparation fermented liquid: 10L, it consists of:

Peptone 200g,

Yeast leach liquor dry powder 100g,

Potassium primary phosphate 20g,

Sodium phosphate dibasic 100g,

Ammonia chloride 50g,

Sodium-chlor 25g,

Add water to 10L,

120 ℃ of sterilizations 30 minutes, be cooled to 37 ℃ after, add respectively glucose 200 gram/300ml and 250mlPWSI after the sterilization ₈₀/ JM103 seed liquor is inserted the B.Braun fermentor tank and is stirred ventilation, regulates with ammoniacal liquor and keeps pH6.8-7.2, fermented 20 hours, reaction finishes, after measured, cell density A600nm 100-150 is with the centrifugal collection thalline of Beckman J6B whizzer 4000rpm weight in wet base 620 grams;

3, the ultrasonic disruption cell

The 620g wet thallus is suspended in 0.05M, in the Tris-HCl damping fluid of pH7.5, ice bath, the ultrasonic disruption cell, 8000rpm is centrifugal with Beckman J2-2M whizzer, and collecting precipitation partly contains 0.1%TweenX-100 with same damping fluid again, 1M NaCl washing 3 times will precipitate lyophilize at last and get 50g fusion rotein dry powder;

4, crack fusion protein

Get the above-mentioned fusion rotein dry powder of 10g, add 3g cyanogen bromide crystal after being dissolved in 300ml 70% formic acid solution, in the dark place under nitrogen covers, stirring at room 2 hours, behind the stopped reaction, concentrating under reduced pressure removes formic acid removal and CNBr, must precipitate, be dissolved in the Tris-HCl damping fluid of pH7.0, the centrifugal insolubles of removing gets Ile ₂₈The about 500mg of-GLP-I (7-36) crude product.

5, use day island proper Tianjin preparation HPLC C ₈High pressure liquid chromatography (HPLC) reversed phase chromatography post 25mm * 50cm, separate pure product Ile ₂₈-GLP-I (7-36) is with WaTers625, HPLC, and 3.9mm * 25cm C4 post, 0.05M Tris-HCl, the pH7.0-30% second cyanogen aqueous solution is that solvent carries out chromatography, tomographic map is the simple spike (see figure 3)

6, show through make conventional biologos mensuration with rat: gained Ile ₂₈-GLP-I (7-36) is compared with natural GLP-I (7-37) has equal short pancreas excreting insulin effect (seeing Fig. 4-7)

Embodiment 2,

The fusion rotein cracking:

Get the above-mentioned fusion rotein dry powder of 10g, be dissolved in 300ml 8M urea, 0.1N HCl, add the CNBr3 gram, in the dark place, nitrogen gas stream covers down, stirring at room 2 hours, behind the stopped reaction, concentrating under reduced pressure is removed after CNBr and the HCl, water-soluble, the centrifugal insolubles of removing is removed urea etc. by the DEAE cellulose chromatography, and wash-out gets Ile ₂₈-GLP-I (7-36) crude product.

All the other operations and product measurement result are with embodiment 1.

Claims (2)

1, a kind of L-iLeu ₂₈-GLUCAGON LIKE PEPTIDE-I (is polypeptide I le ₂₈--GLP-I (7-36)) process for gene engineering production, it is characterized in that:

Adopt A proteolytic enzyme, beta-galactosidase enzymes, the plain CAT of chlorine enzyme, K ₈₈Methionine residues such as antigen protein are that juncture and polypeptide I le28-GLP-I (7-36) form fusion rotein, cut off fusion rotein with CNBr again, obtain product polypeptide I le28-GLP-I (7-36).

2, a kind of L-iLeu according to claim 1 ₂₈-GLUCAGON LIKE PEPTIDE-I the product polypeptide I le28-GLP-I (7-36) of process for gene engineering production gained, it is characterized in that,

Its structure has following form:

His-Ala-Glu-GLY-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-ILe-Gly-Arg-X

But the ester class that X=free radical, free carboxyl group salt, medicine are accepted, an amino acid, different lengths polypeptide, acid amides.

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