Refolding and purification of non-fusion HPT protein expressed in Escherichia coli as inclusion bodies.
The gene encoding hygromycin B phosphotransferase (hpt) is a widely used selectable marker in the production of genetically engineered crops. To facilitate the safety assessment of this protein, the non-fusion hpt expression plasmid was constructed and introduced into Escherichia coli to produce enough quantity of the HPT protein. High level expressed HPT was achieved but most of the expressed protein aggregated as inclusion bodies. The inclusion bodies were washed, separated from the cells, and solubilized by 0.3% Sarkosyl. The protein was renatured by dilution and dialysis, and then purified by anion-exchange chromatography. The activity is 8 U/mg protein and the purity is about 95%. Further studies showed that the microbially produced HPT protein had comparable molecular weight, immuno-reactivities, N-terminal amino acid sequences, and biological activities with those of the HPT produced by transgenic rice harboring hpt gene. All these results demonstrated the validity of utilizing the microbially produced HPT to assess the safety of the HPT protein produced in genetically engineered rice.
Zhuo
Qin
Q
Institute of Nutrition and Food Safety, Chinese Center for Disease Control and Prevention, Beijing 100050, China. zhuoqin1@sina.com.cn
Piao
Jian-hua
JH
Wang
Rui
R
Yang
Xiao-guang
XG
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D005818:Genetic Engineering; D005819:Genetic Markers; D002479:Inclusion Bodies / Q000201:enzymology; D012275:Oryza / Q000201:enzymology / Q000235:genetics; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D030821:Plants, Genetically Modified; D010957:Plasmids / Q000235:genetics; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility
D005819:Genetic Markers; D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase