Method of preparing recombinant small molecular protein or polypeptide with hirudin as fusion partner

A small molecule protein and fusion partner technology, applied in the field of recombinant polypeptide fusion expression technology, can solve the problems affecting the final yield of small molecule target protein and low ratio of small molecule target protein

Inactive Publication Date: 2014-05-28
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] 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

Method used

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  • Method of preparing recombinant small molecular protein or polypeptide with hirudin as fusion partner
  • Method of preparing recombinant small molecular protein or polypeptide with hirudin as fusion partner
  • Method of preparing recombinant small molecular protein or polypeptide with hirudin as fusion partner

Examples

Experimental program
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Embodiment 1

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

[0022] Hirudin III (HV3) fusion tag and target polypeptide Lunasin (Lunasin) fusion protein includes (from N-terminal to C-terminal): (1) 66 amino acid residues of hirudin III (HV3); (2) -GGGDDDDK- Connecting peptide (where DDDDK is the cleavage site of enterokinase); (3) 43 amino acid residues of Lunasin. 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-Lunasin 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 pTASHL (see figure 2 ), and sequenced to verify its correctness. with CaCl 2 The recombinant expression plasmid pTASHL was transformed into E.coli JM109 host bacteria to obtain rHV3-Lunasin fusion protein expression engineering bacteria JM109 / pTASHL.

Embodiment 3

[0025] Example 3 Expression of Hirudin III-Lunasin Fusion Protein in 7L Reactor

[0026]Inoculate a single colony of JM109 / pTASHL 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), 37 ° C stirring culture, the pH of the fermentation broth is controlled at 6.5-7.2 with phosphoric acid, and the dissolved oxygen is 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 feeding 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 fermentation process...

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Abstract

The invention provides a new method of fusion expressing a small molecular protein (polypeptide). The new method is characterized by comprising the following steps: using hirudin as a fusion partner (label), splicing the protein (polypeptide) with a small molecular mesh to the downstream of the hirudin as the fusion partner to carry out fusion expression, designing a connecting peptide (which contains protease or a chemical cutting site or intein as a self-cuttable protein intron) between the fusion partner and a target protein (polypeptide), and releasing the target protein (polypeptide) by restriction enzyme digestion or chemical cutting or induced self cutting after fusion protein expression. The new method has the advantages that (1) as the hirudin as the fusion partner (label) is smaller (with the molecular weight of 7Kd), the rate of the protein (polypeptide) with the small molecular mesh accounting for a fusion protein can be effectively increased, and the yield of the target small molecular protein (polypeptide) is finally increased; (2) the hirudin still has the anticoagulant activity after being fused as the fusion partner (label), and the expression and the purification of the fusion protein can be conveniently detected and traced in real time.

Description

technical field [0001] The invention belongs to the field of genetic engineering and relates to the establishment of a new technology for fusion expression of recombinant polypeptides. The technical feature of the technology is to prepare recombinant small molecule proteins (polypeptides) using hirudin as a fusion partner (label). technical background [0002] When using genetic engineering technology to express some exogenous polypeptides or small molecular proteins, the expression products are often degraded by proteases in the host cells, resulting in a greatly reduced expression. In order to effectively solve this problem, fusion expression technology is widely used at present. The so-called fusion expression refers to splicing exogenous small molecular protein (polypeptide) downstream of the fusion partner (tag) for expression through gene recombination technology, and then releases the target small molecular protein (polypeptide) through enzymatic or chemical cleavage....

Claims

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Application Information

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IPC IPC(8): C07K14/415
CPCC07K14/415C07K2319/35
Inventor 谭树华张萍代广知徐振雷
Owner CHINA PHARM UNIV
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