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Method for heterologously expressing active membrane proteins by using Pichia pastoris expression system

A protein and myosin technology, applied in the fields of genetic engineering and enzymes, can solve problems such as difficulties in the expression of membrane protein activity

Inactive Publication Date: 2013-01-16
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The purpose of the present invention is to provide a method for recombinant active expression of myosin cross-reactivity (MCRA) from Bifidobacterium animalis BB-12 according to the difficulty in the active expression of existing membrane proteins. The method is described in PichiaPink TM Expression System of Membrane Protein MCRA of Bifidobacterium animalis BB-12

Method used

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  • Method for heterologously expressing active membrane proteins by using Pichia pastoris expression system
  • Method for heterologously expressing active membrane proteins by using Pichia pastoris expression system
  • Method for heterologously expressing active membrane proteins by using Pichia pastoris expression system

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Embodiment 1

[0029] PichiaPink transfected with the MCRA gene of Bifidobacterium animalis BB-12 TM Establishment of recombinant bacteria.

[0030](1) Test method: Using the genome of Bifidobacterium animalis BB-12 as a template, design primers according to the MCRA sequence of Bifidobacterium animalis BB-12 (GenBank: ADC85468), and use high-fidelity KOD plus enzyme for PCR amplification of MCRA Gene. Using 5'-CCGGATATCATGGACACTAGGGCGCCGAAAGTCG-3', 5'-CGGGGTACCTCAGTGATGGTGATGGTGATGTTTCGCCGAATCATTCTCCCCCG-3' as primers, the MCRA gene was amplified by high-fidelity PCR. The PCR program is: 95°C for 30s, 55°C for 30s, 68°C for 2.5min, 30 cycles. PCR reaction system: 5 μL dNTPs (2mM), 5 μL 10×KOD plus Buffer, 1 μL KOD plus, 2.5 μL MgSO 4 (25mM), 1 μL of upstream and downstream primers, 2 μL of template. The amplified PCR product was digested with EcoR V and Kpn I at 37°C for 3 h, and then ligated with the pPinkα-HC plasmid digested with Stu I and Kpn I at an appropriate ratio. The ligation...

Embodiment 2

[0034] 1 MCRA gene of Bifidobacterium animalis BB-12 in PichiaPink TM Confirmation of expression in recombinant bacteria.

[0035] 1.1 Experimental method: After the recombinant bacteria and control bacteria were induced by methanol, SDS-PAGE and Wester Blot tests were carried out on the proteins of the bacteria and the culture medium.

[0036] (1) Solution preparation:

[0037] BMGY medium: 1% yeast extract, 2% peptone, 100mM potassium phosphate buffer pH6.0, 1.34% YNB, 0.0004% biotin, 1% glycerol; replace glycerol in BMGY with 1% methanol to obtain BMMY culture base;

[0038] (2) The recombinant bacteria and the control bacteria were cultured in BMGY medium at 28°C and 250rpm for 2 days, then centrifuged at 1500g for 5min, removed the BMGY medium, resuspended the bacteria with BMMY medium, and induced culture at 28°C and 250rpm for 2 days. Two days later, the fermentation broth and cells were taken for SDS-PAGE and Western Blot analysis, and the concentration of the separ...

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Abstract

The invention discloses a method for heterologously expressing active membrane proteins by using a Pichia pastoris expression system. The invention relates to a method for expressing a myosin cross-reactive antigen (MCRA) of Bifidobacterium BB-12 in a recombinant mode and an expression host used in the method. The expression host is a PichiaPinkTM expression system transformed from pPink alpha-HC-MCRA plasmid. The invention also relates to application of an MCRA of Bifidobacterium BB-12 or an expression host in preparing conjugated linoleic acid.

Description

【Technical field】 [0001] The invention relates to the field of genetic engineering and enzyme technology, more specifically, the invention relates to the active expression, enzymatic function and scientific research application of a membrane protein. 【Background technique】 [0002] Pichia pastoris is a yeast strain that can efficiently express recombinant proteins. Compared with other expression systems, the Pichia pastoris expression system has the following advantages: 1) Unique and powerful AOX (alcohol oxidase Gene) promoter, methanol can strictly regulate the expression of exogenous genes, and methanol is cheaper than IPTG, which is generally used for expression induction in Escherichia coli; 2) The expression level is high, which can be expressed in cells or secreted; 3) The fermentation process is mature and easy to scale up; 4) The culture cost is low and the product is easy to separate; 5) The foreign protein gene is genetically stable. Generally, the exogenous pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/31C12N1/19C07K14/195C12P7/64C12R1/84
Inventor 陈海琴陈卫杨波田丰伟赵建新宋元达陈永泉张灏
Owner JIANGNAN UNIV
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