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7 beta-HSDH enzyme and DPS fusion protein

A technology of fusion protein and DPS, applied in the field of fusion protein, can solve the problems of complex process and low yield, etc.

Active Publication Date: 2020-02-11
BIORTUS BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The process of preparing tauroursodeoxycholic acid (TUDCA) by chemical synthesis is complicated and the yield is low. The activity and thermal stability of 7β-HSDH enzyme in the biotransformation method need to be further improved to meet the needs of industrial production

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  • 7 beta-HSDH enzyme and DPS fusion protein
  • 7 beta-HSDH enzyme and DPS fusion protein
  • 7 beta-HSDH enzyme and DPS fusion protein

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Embodiment Construction

[0017] The present invention is described below through specific embodiments. Unless otherwise specified, the technical means described in the present invention are methods known to those skilled in the art. Furthermore, the embodiments should be understood as illustrative, not limiting the scope of the invention. The spirit and scope of the present invention are defined only by the claims.

[0018] 1. Materials

[0019] 1.1 Expression vector: (1) pGEX6P-1-N-GST-3C-7β-HSDH, its plasmid map is shown in 1;

[0020] (2) pGEX6P-1-N-GST-3C-DPS-linker-7β-HSDH was constructed by our company, and its plasmid map is shown in 2.

[0021] 1.2 Competent cells: TransT1 competent cells, BL21 competent cells.

[0022] 1.3 Medium and reagents: LB medium, IPTG inducer, L-Glutathione (Reduced), SYPROOrange dye, NADP, TUDCA, plasmid mini-extraction kit (TIANGEN), ordinary agarose gel recovery kit (TIANGEN)

[0023] 1.4 Experimental equipment SPH-211C temperature-controlled shaker, AKTA-FPLC...

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Abstract

The invention relates to a 7 beta-HSDH enzyme and DPS fusion protein. The 159th glycine of the 7 beta-HSDH enzyme of which an amino acid sequence is as shown in SEQUENCE NO.1 is mutated to form histidine, the 165th lysine is mutated to form histidine, a 7 beta-HSDH enzyme mutant of which an amino acid sequence is as shown in SEQUENCE NO.2 is obtained, the 7 beta-HSDH enzyme mutant and DPS proteinwhich is derived from thermoanaerobacter pseudethanolicus are subjected to fusion expression so as to obtain the 7 beta-HSDH enzyme and DPS fusion protein. Compared with a wild 7 beta-HSDH enzyme, the7 beta-HSDH enzyme and DPS fusion protein obtained by the invention has the advantages that the heat stability is significantly improved, and the enzymic activity is improved by 70%.

Description

technical field [0001] The invention relates to the technical field of fusion proteins, in particular to a fusion protein of 7β-HSDH enzyme and DPS. Background technique [0002] Tauroursodeoxycholic acid (TUDCA Ursodeoxycholic acid) is the main pharmacological active ingredient in bear bile, which is currently mainly obtained by artificially extracting bear bile. Tauroursodeoxycholic acid is mainly used clinically to treat diseases related to abnormal bile acid metabolism. It is far from meeting the clinical drug demand. At present, TUDCA can be prepared not only from bear bile, but also by chemical synthesis and biotransformation. The chemical synthesis method to prepare tauroursodeoxycholic acid (TUDCA) is complicated and the yield is low. The activity and thermal stability of 7β-HSDH enzyme in the biotransformation method need to be further improved to meet the needs of industrial production. Contents of the invention [0003] The present invention analyzes and comp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/70
CPCC07K2319/00C12N9/0006C12N15/70C12Y101/01201
Inventor 王峰吕志佳徐霞陈倩缪奇微
Owner BIORTUS BIOSCI
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