[Expression of von willebrand factor-A3 domain in E coli and its biological function].
The interaction among collagen, von Willebrand factor (vWF) and glycoprotein Ib axis is the first step in hemostasis and thrombosis, especially under high shear condition. To develop a new remedy of anti-thrombosis, mRNA from endothelial cells was extracted, and reverse transcription PCR was adopted to amplify DNA of interest. After sequencing, recombinant expression vector was constructed. The amplified DNA fragment of vWF domain A3 was inserted into expression vector with 6 x his taq, pET20b(+), the recombinant was transformed into E coli (strain DE3) and induced by IPTG. Recombinant vWF-A3 was designated as a recombinant fragment comprising residues 918 - 1114 of mature vWF subunit. It was purified through Ni-NTA resin column and refolded in Tris buffer containing GSH and GSSG. The results showed that rvWF-A3 was expressed successfully in E coli (strain DE3), accounting for 46% of total bacterial protein with its purity of over 95%. It was identified that rvWF-A3 is capable to bind collagen and inhibit the wild vWF binding to collagen by competition. It is concluded that rvWF-A3 fragment might be an effective antithrombotic agent for preventing arterial thrombosis.
Zhu
Huai-Ping
HP
Jiangsu Institute of Hematology, The First Affiliated Hospital of Suzhou University, Suzhou 215006, China.
Wang
Ying-Chun
YC
Ji
Shun-Dong
SD
Bai
Xia
X
Jiang
Miao
M
Ruan
Chang-Geng
CG
D003001:Cloning, Molecular; D003094:Collagen / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014841:von Willebrand Factor / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D011994:Recombinant Proteins; D014841:von Willebrand Factor; D003094:Collagen