[Non-fusion expression and purification of cowpea typsine inhibitor].
OBJECTIVE
To establish a practicable method to express and purify Cowpea Typsine Inhibitor (CpTI), which is comparable with native CpTI, for providing important materials to access the safety of genetically modified rice with CpTI.


METHODS
The non-fusion expression vector PET41EK-CpTI was constructed by cloning the CpTI gene into expression vector PET41EK. And the expression vector was then transformed into E. coli BL21 (DE3) and E. coli Rosetta ( DE3). Purified expression product was obtained through optimizing the induction temperature, induction time and IPTG concentration and then it was identified by western blotting, activity determination and N-terminal amino acid sequencing.


RESULTS
The purified protein was obtained under the condition of E. coli Rosetta (DE3), 20 - 25 degrees C, 130 rpm, and 0.5 mmol/L IPTG, overnight expression, 30kDa ultra-filtration and anion exchange purification. After identification, the CpTI protein with significant immunogenicity and CpTI activity was successfully obtained and its purity is up to 95%.


CONCLUSIONS
The CpTI protein obtained by the non-fusion method established in this study is comparable with the native CpTI protein in terms of activity, immunogenicity, molecular weight and amino acids sequence. Moreover, the method is easy to operate and satisfied with the requirements of large-scale expression.
The Key Laboratory of Trace Element Nutrition, Ministry of Health of the People's Republic of China.
Yichun
Y
Hu
Qin
Q
Zhuo
Jianhua
J
Piao
Xiaoguang
X
Yang
D015153:Blotting, Western; D002846:Chromatography, Affinity; D004926:Escherichia coli; D007887:Fabaceae / Q000737:chemistry*; D014361:Trypsin Inhibitors / Q000302:isolation & purification*
D014361:Trypsin Inhibitors