Novel method for preparing recombinant exenatide or derivative thereof

A technology for exenatide and its derivatives, which is applied in the field of preparing exenatide or its derivatives with natural N-terminals, which can solve the problems of low ratio of small molecule target protein, affecting the final yield of small molecule target protein, etc. , to achieve the effect of enhancing stability, increasing the final expression level, and avoiding degradation

Inactive Publication Date: 2015-09-09
CHINA PHARM UNIV
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, when these fusion partners (tags) are used for fusion expression of small molecular target proteins (polypeptides), although fusion proteins are easily expressed at a high level, due to the large molecular weight of the fusion partners (tags), the small molecular target protein The ratio of (polypeptide) in the fusion protein is relatively low (usually only 1 / 5 to 1 / 10 or even lower of the fusion protein), which will inevitably seriously affect the final yield of the small molecule target protein (polypeptide). Rate

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  • Novel method for preparing recombinant exenatide or derivative thereof
  • Novel method for preparing recombinant exenatide or derivative thereof
  • Novel method for preparing recombinant exenatide or derivative thereof

Examples

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Embodiment 1

[0021] Example 1 Design and Cloning of Hirudin III (HV3) Fusion Tag and Target Polypeptide Exendin (Exendin-4) Fusion Gene

[0022]Hirudin III (HV3)-Exendin-4 fusion protein includes the following parts (from N-terminal to C-terminal): (1) 66 amino acid residues of hirudin III (HV3); (2) connecting peptide GGGGSENLYFQ↓H (where ENLYFQH is the TEV enzyme recognition cleavage sequence, the arrow indicates the cleavage site); (3) TEV enzyme cleavage site (indicated by the arrow) is followed by 39 amino acid residues of Exendin-4 base. On this basis, the above-mentioned fusion protein encoding gene was designed and synthesized according to E.coli preferred codons (see figure 1 ).

Embodiment 2

[0023] Example 2 Construction of Hirudin III (HV3)-Exendin (Exendin-4) Fusion Protein Expression Strain

[0024] The fusion gene described in Example 1 was digested with Nhe I and Hind III and then subcloned into the expression vector pTASH (Tan S, Wu W, Liu J, Protein Expr Purif2002, 25, 430-436.) site, the resulting recombinant expression vector was named pTASHE (see figure 2 ), and sequenced to verify its correctness. with CaCl 2 The recombinant expression plasmid pTASHE was transformed into E.coli JM109 host strain to obtain rHV3-Exendin-4 fusion protein expression engineered strain pTASHE / JM109.

Embodiment 3

[0025] Example 3 Expression of Hirudin III (HV3)-Exendin (Exendin-4) Fusion Protein in 7L Reactor

[0026] Inoculate a single colony of pTASHE / JM109 engineering bacteria in 200ml LB liquid medium (containing 100μg / ml ampicillin) at 37°C, 220rpm, and culture for 12h. Inoculate 5L fermentation medium (1% tryptone, 0.5% yeast powder, 4% sodium glutamate, 1% malt Powder, 0.671%KH 2 PO 4 , 0.757% Na 2 HPO 4 12H2O, 100 μg / ml ampicillin, pH 6.5), stirred culture at 37°C, the pH of the fermentation broth was controlled at 6.5-7.2 with phosphoric acid, and the dissolved oxygen was controlled at 40%-60%. When the fermentation broth OD 600nm When reaching 3.0 with about 30ml h -1 l -1 Feed medium I (10% malt powder, pH 6.5), the feed time is maintained for about 4 hours and the growth of the bacteria reaches the plateau stage. -1 l -1 Feed medium II (3.33% peptone, 1.67% yeast powder, 13.3% sodium glutamate, 10% malt powder, pH6.5) until the end of fermentation. The whole ferme...

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Abstract

The invention builds up a novel method for preparing recombinant exenatide or derivative thereof. According to the method, hirudin (molecular weight is 7Kd) serves as a fusion partner, exenatide or derivative thereof is spiced on the downstream of the hirudin for fusion expression, a connecting peptide is arranged between the hirudin fusion partner and the exenatide or derivative thereof and comprises a TEV protease (tobacco etching virus protease) identification cutting sequence ENLYFQH. The exenatide or derivative thereof with a natural N-terminal and biological activity is released through TEV enzyme digestion after fusion protein expression. The novel method has other advantages that (1) the hirudin fusion partner is small, so the ratio of the exenatide or derivative thereof in fusion protein is effectively increased; (2) hirudin fusion protein has anticoagulation activity, thus facilitating real-time detection and tracing; (3) fusion protein can be adsorbed by virtue of cheap macroporpous resin; and (4) hirudin fusion partner can enhance stability of exenatide or derivative thereof.

Description

technical field [0001] The invention belongs to the field of genetic engineering pharmacy and relates to establishing a new method for preparing exenatide or its derivatives with natural N-terminal. The technical characteristics of the method are: using hirudin as a fusion partner (label) to prepare recombinant exenatide or its derivatives, and designing a connection between the fusion partner hirudin and the target product exenatide or its derivatives Peptide, the connecting peptide contains TEV enzyme (tobacco etch virus protease) recognition cleavage sequence ENLYFQH, so that after the fusion protein is expressed and purified, the N-terminus can be released by TEV digestion, which is completely consistent with wild-type exenatide and has biological activity The target product exenatide. technical background [0002] Diabetes mellitus (DM) is a complex metabolic disease in which blood sugar rises due to endocrine and metabolic disorders caused by insufficient insulin secr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C07K14/575
Inventor 谭树华张苗青韩宁
Owner CHINA PHARM UNIV
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