[Expression and identification of recombinant P-selectin and P-selectin glycoprotein ligand-1].
P-selectin, one of the membrane proteins, expresses on platelet and endothelia and interacts with P-selectin glycoprotein ligand-1 (PSGL-1) on leukocyte membrane. This interaction mediates leukocytes rolling on endothelial membrane and then induces leukocyte recruitment to the site of infection or tissue injury. In the present study, we constructed the recombinant wild type human P-selectin, its calcium-binding sites mutants and recombinant PSGL-1-globulin (PSGL-1-Rg). They expressed in Sf9 cells by using the baculovirus expression system and were purified by TalonTM metal or Protein A affinity chromatography. The results showed that the recombinant PSGL-1-Rg interacted with recombinant wild type P-selectin and two P-selectin mutants with 2 calcium-binding sites mutation respectively, but could not bind to the P-selectin mutant with all 4 calcium-binding sites mutation. Therefore, we verified the importance of P-selectin calcium-binding sites for its interaction with PSGL-1.
Pei
Xin-Hui
XH
College of Life Science and Biotechnology, Shanghai Jiaotong University, Shanghai 200240, China.
Lin
Zhi-Xin
ZX
Geng
Jian-Guo
JG
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D006801:Humans; D007962:Leukocytes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D009154:Mutation; D019007:P-Selectin / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D008562:Membrane Glycoproteins; D019007:P-Selectin; C084882:P-selectin ligand protein; D011994:Recombinant Proteins; C546937:SELP protein, human; D002118:Calcium