Construction of spider draggling silk protein MaSp1 prokaryotic expression vector and its expression and purification in Escherichia coli
10.7644/j.issn.1674-9960.2014.08.013
- VernacularTitle:蜘蛛牵引丝蛋白MaSp1原核表达载体构建及其在大肠杆菌中的表达与纯化
- Author:
Xin QIAO
;
Yan WANG
;
Junjie LI
;
Cuimi DUAN
;
Haibin WANG
;
Jin ZHOU
;
Zhiyan DU
;
Changyong WANG
- Publication Type:Journal Article
- Keywords:
MaSp1;
genetic engineering;
prokaryotic expression;
protein purification
- From:
Military Medical Sciences
2014;(8):621-625
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a key technological system for spider fibroin gene code tandem connection , vector construction , prokaryotic expression and purification using genetic engineering in order to achieve MaSp 1 heterologous ex-pression in Escherichia coli and its separation and purification .Methods Isocaudarner ligation method was used to connect synthetic spider fibroin gene monomer code in tandem , and a recombinant clone concatemer was obtained .The identified recombinant clones were connected with prokaryotic expression vector pET 28a(+), and then transformed into E.coli BL21 (DE3).After being induced by IPTG for 6 hours, the expression product was identified by SDS-PAGE and Western blot-ting.Engineering bacteria were fermented in high density , and the obtained protein was purified through ammonium sulfate fractionation.Results and Conclusion The expression plasmids of MaSp1concatemers were successfully constructed , and the induced expression genetic engineering MaSp 1 protein was of the expected relative molecular mass .In addition, the pu-rity of the purified protein was above 80%.This study has developed crucial technologies for mass production of genetic en-gineering spider silk proteins .
