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Prokaryotic expression vector contributing to purification of foreign protein and construction method thereof

A prokaryotic expression and construction method technology, applied in the field of genetic engineering, can solve problems such as high price affinity chromatography column

Inactive Publication Date: 2011-06-01
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method is easy to operate, it still requires the use of expensive affinity chromatography columns, which pushes the production cost of recombinant proteins to a certain height

Method used

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  • Prokaryotic expression vector contributing to purification of foreign protein and construction method thereof
  • Prokaryotic expression vector contributing to purification of foreign protein and construction method thereof
  • Prokaryotic expression vector contributing to purification of foreign protein and construction method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0035]Example 1: Construction of pET-EM

[0036] The construction process of pET-EM is as follows figure 2 ,Specific steps are as follows:

[0037] 1) with restriction endonuclease SAP I and Bgl I digest the vector pET30a, separate the digested products by electrophoresis on 0.8% agarose gel, recover the large fragments digested by the DNA recovery kit in the gel, fill in the ends of the recovered products with Klenow large fragment DNA polymerase, and connect them with T4 DNA ligase. get none SAP The vector pET30a' of I restriction site;

[0038] 2) According to the Mxe on the commercial vector pYTB11 gyrA Intein coding sequence, design and synthesize a pair of primers:

[0039] P1: 5'-TCCATATGGGTACCCCATGGGCTAGCTCTAGAGGCTCTTCCTGCA

[0040] TCACGGGA-3' (SEQ ID NO.1) (introduced at the 5' end of the primer Nde I. Kpn I. Nco I. Nhe I. Xba I. SAP I restriction site);

[0041] P2: 5'-TT GATATC AGAGCGTGGCTGACGAACCCG-3' (SEQ ID NO.2) (the underline is intr...

Embodiment 2

[0044] Example 2: Prokaryotic expression and purification of African swine fever virus K205R gene

[0045] 1. Synthesize a pair of primers P1: 5'-GGTCATATGGTTGAGCCACGCGAACAG-3' (SEQ ID NO.3), P2: 5'-GGTTGCTCTTCCGCACTTCTTCATCATCTCTTTG-3' (SEQ ID NO.4), PCR method, from plasmid pGEX-4T-1 -K205R amplifies the K205R gene ( image 3 ), to recover the DNA fragment, the restriction enzyme Nde I and SAP Ⅰ After double digestion, insert the vector pET-EM to construct the recombinant expression vector pET-EM-K205R ( Figure 4 ).

[0046] 2. Transform the recombinant expression vector pET-EM-K205R into Escherichia coli BLR (DE3) competent cells, screen out positive recombinant bacteria, inoculate 50ug / ml kanamycin LB liquid medium, and place in a shaker (160rpm) at 37°C When the culture OD600 is 0.5, add IPTG to the final concentration of 0.4mM, induce at 20°C for 24h, precipitate the bacteria, and freeze repeatedly in 0.5ml lysis buffer (10mM Tris-HCl, 2mM EDTA, 0.1mg / ml Lysozyme,...

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Abstract

The invention relates to a prokaryotic expression vector contributing to the purification of a foreign protein and a construction method thereof. The structure of the prokaryotic expression vector is shown by a figure 1. The prokaryotic expression vector is constructed by inserting a multiple cloning site and an Mxe-ELP gene sequence on the basis of a carrier pET 30a. A foreign protein coding gene to be expressed is inserted into the multiple cloning site to convert competent host cells; and after induced expression, the expressed foreign protein is separated and purified on the basis of the biological characteristics of elastin-like polypeptides and the characteristic that inten MxeGyrA can perform self severing in the presence of thiol. Compared with other prokaryotic expression system, the expression system avoids using expensive chromatographic technique, makes operation simple and easy, has a very obvious advantage in the field of protein production, and is very suitable for the expression and purification of soluble proteins.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a construction method and application demonstration of a prokaryotic expression vector that facilitates the expression and purification of exogenous proteins. Background technique [0002] The use of prokaryotic expression system to express some specific proteins in vitro is one of the commonly used protein production methods. Its biggest advantages are high protein yield and easy operation. At present, there are many mature prokaryotic expression systems. Traditional recombinant protein expression and purification usually add specific tags (such as GST tag, His tag, etc.) before or after the target protein. The fusion protein formed can be used for affinity chromatography with the help of protein tags. The cost of protein purification is high, and the purified fusion protein often needs to be cleaved in vitro to remove the protein tag to obtain the target protein wit...

Claims

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Application Information

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IPC IPC(8): C12N15/66
Inventor 孙怀昌朱善元张鑫宇王健刘文俊夏晓莉
Owner YANGZHOU UNIV
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