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Colibacillus expression carrier and its application

An expression vector, a technology of Escherichia coli, applied in the field of bioengineering, can solve the problems of low yield and high cost of 7-aminocephalosporanic acid

Active Publication Date: 2007-02-14
常熟盈赛生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thereby enzymatic method prepares 7-amino cephalosporanic acid at present and has the shortcoming that cost is high, productive rate is low

Method used

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  • Colibacillus expression carrier and its application
  • Colibacillus expression carrier and its application
  • Colibacillus expression carrier and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Construction of expression vector pRSET-kan

[0019] Design PCR primers according to the sequence of pRSET-A, specifically:

[0020] Upstream primer VET-F: 5’-CTGTCAGACCAAGTTTACTCATATATACTTTAG-3’;

[0021] Downstream primer VET-R: 5'-ACTCTTCCTTTTTCAATATTATTGAAGC-3'.

[0022] The PCR primers were designed according to the sequence of pET28b, specifically the upstream primer KAN-F: 5'-ATGAGCCATATTCAACGGGAAAC-3'; the downstream primer KAN-R: 5'-TTAGAAAAACTCATCGAGCATCAAATG-3'.

[0023] The PCR conditions for amplifying the pRSET-A fragment with the ampicillin resistance gene removed are: 50ng pRSET-A (Invitrogen), 0.4μM VET-F, 0.4μM VET-R, 50μM dATP, 50μM dTTP, 50μM dCTP, 50μM dGTP, 20mM Tris-HCl (pH8.8), 10mM KCl, 10mM (NH 4 ) 2 SO 4 , 2mM MgSO 4 , 0.1% Triton X-100, 2.5U Pfu DNA polymerase (Promega), adjust the reaction volume to 50 μL with sterile water. The PCR amplification reaction program is: 94°C, 5 minutes; 94°C, 1 minute, 50°C, 1 minute, 72°C, 4 minutes, cy...

Embodiment 2

[0026] Example 2: Construction of vector pRSET-lac-kan

[0027] Design PCR primers according to the sequence of pGEMT-Easy (Promega), specifically: upstream primer RBS-NdeI: 5’- CATATG TATATC TGTGTGAAATTG-3' (the NdeI restriction site is underlined, and the additional ribosome binding site is represented by the dashed line below); downstream primer RBS-AlwNI: 5’- CAGTGGCTG CTGCCAGTGGCGATAAGTC-3' (AlwNI restriction site is underlined).

[0028]Using pGEMT-Easy (Promega) as a template, PCR was performed with the above primers, and a 755bp product was amplified. The PCR conditions were: 50ng pGEMT-Easy (Promega), 0.4μMRBS-NdeI, 0.4μM RBS-AlwNI, 50μM dATP, 50μM dTTP, 50μM dCTP, 50μM dGTP, 20mM Tris-HCl (pH 8.8), 10mM KCl, 10mM (NH 4 ) 2 SO 4 , 2mM MgSO 4 , 0.1% Triton X-100, 2.5U Pfu DNA polymerase (Promega), adjust the reaction volume to 50 μL with sterile water. The PCR amplification reaction program is: 94°C, 5 minutes; 94°C, 1 minute, 50°C, 1 minute, 72°C, 4 minutes, cycle 35 t...

Embodiment 3

[0031] Example 3: Construction of vector pGEMT-Easy-GI

[0032] According to the known Thermoanaerobacterium saccharolyticum glucoseisomerase DNA sequence (GenBank L09699), design PCR primers, specifically: upstream primer (GI-NdeI):

[0033] 5’- CATATG AATAAATATTTTGAGAACGTATCTAAAATA-3' (NdeI restriction site is underlined);

[0034] Downstream primer (GI-EcoRI): 5’- GATATC TTAA TTATTCTGCAAAC-3' (EcoRI restriction site is underlined, AscI restriction site is double-underlined).

[0035] Using Thermoanaerobacterium saccharolyticum (purchased from ATCC, USA) DNA as a template, PCR was performed with the above primers, and a 1,336bp product was amplified. PCR conditions were: 50ng T.saccharolyticum DNA, 0.4μM GI-NdeI, 0.4μM GI-EcoRI, 50μM dATP, 50μM dTTP, 50μM dCTP, 50μM dGTP, 20mM Tris-HCl (pH 8.8), 10mM KCl, 10mM (NH 4 ) 2 SO 4 , 2mM MgSO 4 , 0.1% Triton X-100, 2.5U Platinum Taq High Fidelity DNA polymerase (Invitrogen), adjust the reaction volume to 50 μL with sterile water. Th...

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Abstract

The invention provides Escherichia coli expression vector and its using method. The expression vector is formed by vector pRSET-A whose ampicillin resistant gene is replaced by kanamycin resistant gene. And it can be used to express the enzyme needed in making 7-aminocephalosporanic acid but no beta-lactamase reduced its yield. The invention also provides two concrete expression vectors that pHS-GHA and pT7-kan-ACY whose gene products are respectively D-amino acid oxidase mutant GHA and Pseudomonas sp.SE83 glutaryl-7-aminocephalosporanic acid acidylating enzyme.

Description

Technical field [0001] The present invention relates to the technical field of bioengineering. Specifically, the present invention relates to an Escherichia coli expression vector that does not produce β-lactamase. The expression vector can be used for high-efficiency expression in the preparation of 7-aminocephalosporin Enzymes required for acid. Background technique [0002] Enzymatic preparation of 7-aminocephalosporanic acid involves two steps: (1) D-amino acid oxidase oxidizes cephalosporin C to produce glutaryl-7-aminocephalosporanic acid; (2) glutaryl-7- Aminocephalosporanic acid acylase hydrolyzes glutaryl-7-aminocephalosporanic acid to produce 7-aminocephalosporanic acid. At present, most of the expression vectors for the above two enzymes use ampicillin resistance genes as selection markers. But its gene product β-lactamase, in addition to degrading ampicillin, it also degrades β-lactam compounds cephalosporin C, glutaryl-7-aminocephalosporanic acid and 7-aminocephalosp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/53C12P21/02
CPCC12N15/70C12P21/02
Inventor 王骏曾伟基叶康坚刘兆明肖游龙曾实现
Owner 常熟盈赛生物科技有限公司
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