[Expression of platelet collagen receptor-glycoprotein VI fragment in E. coli and its biological activities].
This study was aimed to further investigate the function of platelet collagen receptor-glycoprotein VI and to screen its specific inhibitor. The extracellular domain of platelet glycoprotein VI (GPVI) in E. coli was expressed by recombinant technology, the extracellular domain cDNA of GPVI was amplified from pBluescript KS(-)-GPVI plasmid by PCR. Proved by sequencing, the expression vector pET-20b(+)-GPVI was constructed, which was then transformed into E. coli (BL21(DE3)pLysS) and induced by IPTG. The recombinant GPVI was purified on Ni-NTA resin column and renatured in PBS containing GSH and GSSG. The anti-penta His McAb and anti-GPVI polyclonal antibody were used to identify the recombinant GPVI in Western blotting. Collagen binding test was conducted to investigate the biological activity of recombinant GPVI. The results showed that the recombinant GPVI was expressed in E. coli and successfully purified, which was confirmed to be similar to the native GPVI in Western blotting. The recombinant GPVI can bind the type I collagen in dose-dependent manner. In conclusion, the recombinant GPVI can be achieved in E. coli and restore its native characteristics after renaturation.
Yu
Zi-Qiang
ZQ
The First Affiliated Hospital of Suzhou University, Jiangsu Institute of Hematology, Suzhou 215006, China.
Dong
Ning-Zheng
NZ
Bai
Xia
X
Zhu
Huai-Ping
HP
Ji
Shun-Dong
SD
Jiang
Miao
M
Ruan
Chang-Geng
CG
D001792:Blood Platelets / Q000378:metabolism*; D015153:Blotting, Western; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D038982:Integrin alpha2beta1; D010980:Platelet Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D011485:Protein Binding; D038981:Receptors, Collagen / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
D038982:Integrin alpha2beta1; D010980:Platelet Membrane Glycoproteins; D038981:Receptors, Collagen; D011994:Recombinant Proteins; C098341:platelet membrane glycoprotein VI; C468113:type I collagen receptor