[Study on vector construction, expression and refolding of fusion HPT protein].
OBJECTIVE
Hygromycin B Phosphotransferase gene (hpt) is a widely used antibiotic selectable marker gene in the production of genetically engineered crops. Fusion expression vector of the hpt gene was constructed in order to obtain enough HPT protein for further safety assessment of the protein.


METHODS
The hpt gene was cloned into liner expression vector pET41 EK/LIC and fusion expression vector pET 41EK/HPT was obtained. The constructed vector was transformed into expression host E. coli BL21 (DE3). After induction, the expressed fusion protein was mainly aggregated as inclusion bodies. To get the active protein, dilution refolding, chromatographic refolding and Sarkosyl resolution and dilution refolding methods were explored. Then the fusion protein was digested by enterokinase and N-terminal amino acid sequence was conducted.


RESULTS
The refolding study showed the protein got from Sarkosyl resolution and dilution refolding method had higher yield and bioactivity than those from other two methods. From this method 78mg active fusion obtained protein could be obtain from 1L bacterial culture and the purity was about 93%. From N terminal amino acid sequence it was confirmed that after digested by enterokinase, the HPT protein had same N-terminal amino acid sequence as the native HPT.


CONCLUSIONS
The results of this study illustrated that large quantity HPT protein could be acquired through prokaryotic host expression which had comparable molecular weight, N-terminal amino acid sequences and biological activities with those of native HPT protein.
Zhuo
Qin
Q
Institute for Nutrition and Food Safety, Chinese Center for Disease Control and Prevention, Beijing 100050, China. xgyangcdc@vip.sins.com
Tian
Yuan
Y
Wang
Rui
R
Piao
Jianhua
J
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005819:Genetic Markers; D005822:Genetic Vectors; D012275:Oryza / Q000235:genetics; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis* / Q000235:genetics; D030821:Plants, Genetically Modified; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics
D005819:Genetic Markers; D011993:Recombinant Fusion Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase