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Oriented mutant gene engineering barr kinase and its use

A directed mutation and genetic engineering technology, applied in the field of batroxobin, can solve problems such as proteins with complex sugar chains in space

Active Publication Date: 2007-04-04
GRAND LIFE SCI (LIAONING) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, proteins with complex spatial structures and sugar chains are not suitable for expression in prokaryotic cells

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066]According to the nucleic acid sequence of batroxobin (X12747) disclosed in American Genebank, the genetic codes preferred by yeast were selected, the structural gene sequence of batroxobin was artificially synthesized, and the ten amino acids outside the C-terminal of batroxobin were deleted directionally. , and also mutated Tyr at position 133 of the active center site to Glu to enhance the negative potential of the negative potential region of the active center, thereby enhancing the electrostatic interaction between the enzyme and the substrate. In order to insert the synthetic target gene into the yeast secretion expression vectors pPIC9K and pPICZα, add an Xho I restriction site at the 5' end of the gene, and add a stop codon TAATGA and a Not I restriction site at the 3' end of the gene . In addition, the codon AAAAGA corresponding to the KEX2 protease recognition sequence Lys-Arg is added between the Xho I restriction site and the first amino acid Val codon at the ...

Embodiment 2

[0069] Linearize pPIC9K-Bg with DNA restriction endonuclease SacI, prepare yeast host competent and transform according to the method in Multi-Copy Pichia Expression Kit Version F of Invitrogen Company, and spread the transformed cells on RDB medium for growth , cultured at 30°C, a single colony can appear after 4-6 days. Detect the number of copies recombined into the yeast genome on YPD plates with different concentrations of Geneticin: YPD+Geneticin (0-0.25mg / ml) is used to detect host bacteria containing one copy number; YPD+Geneticin (0.5-4mg / ml) is used to detect host bacteria with more than two copies. Cultivate at 30°C, and a single colony will appear after 2-5 days, pick a single colony and streak culture, and save the strain.

[0070] Linearize pPICZa-Bg with DNA restriction endonuclease SacI, prepare yeast host competent and transform according to the method in Multi-Copy Pichia Expression Kit Version B of Invitrogen Company, and spread the transformed cells on YP...

Embodiment 3

[0072] The high-expression engineered bacteria screened in test tubes were inoculated into shake flasks for proliferation as seed liquid, and then transferred to a 30L fermenter for pilot-scale fermentation according to the conventional method of methanol yeast fermentation. Induction for 50 hours. The supernatant is collected by centrifugation of the fermentation broth, or concentrated and desalted by ultrafiltration, and biochemical separation techniques such as hydrophobic column chromatography, ion exchange column chromatography, affinity column chromatography, gel column chromatography, etc. are used to obtain the purity of more than 97%. active protein.

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Abstract

A directed mutation gene engineering batroxobin and its use are disclosed. The process is carried out by deleting 10 amino acids at C-end of batroxobin amino-acid sequence and mutating No.133 Tyr to Glu. It has better biological activity.

Description

Technical field: [0001] The invention relates to a gene recombination method for preparing directed mutation batroxobin—Batroxobin. The key is the directional transformation, synthesis, cloning, expression of the batroxobin gene and the fermentation expression and separation and purification of the product. The mutated batroxobin can be used not only as the main component of the hemostatic drug, but also directly as the hemostatic drug. Background technique: [0002] Since 1963, the Austrian scholar Von Klobusitzky isolated a serine proteolytic enzyme (thrombin-like enzyme) from the venom of the Brazilian spearhead viper (Bothropsatrox) for the first time, also known as batroxobin. So far, more than 30 kinds of snake venoms have been found to contain thrombin-like components, and more than 20 kinds have been isolated and purified successively. They can all act on Arg in the A chain of fibrinogen in mammalian plasma. 16 -Gly 17 The peptide bond between them releases fibrin...

Claims

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Application Information

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IPC IPC(8): C12N9/50C12N9/74C12N15/81C12N15/57C07H21/00A61K38/48A61P7/04
Inventor 薛百忠薛雁王宏英徐梅沈文彧苏珊
Owner GRAND LIFE SCI (LIAONING) CO LTD
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