Prokaryotic expression and biological activity analysis of human arresten gene.
To express recombinant arresten in Escherichia coli (E. Coli) and investigate its biological activity, prokaryotic expression vector of human arresten gene was constructed by gene engineering. Human arresten gene was amplified from recombinant plasmid pGEMArr by polymerase chain reaction (PCR), and inserted into prokaryotic expression vector pRSET containing T7 promoter. Restriction analysis and DNA sequencing verified that the arresten gene was correctly cloned into the expression vector. The recombinant plasmid pRSETAt was subsequently transformed into E. coli BL21 (DE3), and the target gene was expressed under induction of IPTG. SDS-PAGE analysis revealed that the recombinant protein with a molecular weight of 29 kD (1 kD = 0.992 1 ku) amounted to 29% of the total bacterial proteins. After purification and renaturation, the recombinant protein could significantly suppress the proliferation of human umbilical vein endothelial cells (HUVECs). These results suggested that the expression of a biologically active form of human arresten in the pRSET expression system laid a foundation for further study on the mechanistic insight into arresten action on angiogenesis and the development of powerful anti-cancer drugs.
Song
Zifang
Z
Department of General Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Qichang
Zheng
Z
Li
Wei
W
Xiong
Jun
J
Shang
Dan
D
Shu
Xiagogang
X
D020533:Angiogenesis Inhibitors / Q000096:biosynthesis / Q000235:genetics*; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D003001:Cloning, Molecular; D024141:Collagen Type IV / Q000096:biosynthesis / Q000235:genetics*; D004730:Endothelium, Vascular / Q000166:cytology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D011387:Prokaryotic Cells / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection; D014471:Umbilical Veins / Q000166:cytology
D020533:Angiogenesis Inhibitors; D024141:Collagen Type IV; D011994:Recombinant Proteins