One-step affinity purification of recombinant alphavbeta3 integrin from transfected cells.
Vitronectin receptor (alphavbeta3 integrin) is present on the surface of many types of cells. We describe a simple, fast, and reliable method of purification of recombinant human alphavbeta3 from Chinese hamster ovary (CHO) cells transfected with alphavbeta3 (VNRC3 cells). The method consists of two steps: lysis of the cells and affinity chromatography of the lysate on a GRGDSPK-Sepharose column. The yield of the procedure was about 79%. The purified receptor migrated as two bands on a silver stained SDS-polyacrylamide gel, corresponding to the alphav and beta3 subunits, and was recognized by monoclonal antibodies directed against alphav and the alphavbeta3 complex, but not by monoclonal antibody specific for the alphaIIbbeta3 complex. This receptor also bound to immobilized vitronectin, von Willebrand factor, and echistatin. However, binding to immobilized fibrinogen was not observed. Purified recombinant alphavbeta3 demonstrated greater immunoreactivity with LM 609, an alphavbeta3 complex-specific monoclonal antibody, than alphavbeta3 purified from placenta. As visualized by SDS-polyacrylamide gel electrophoresis, preparations of placenta-derived alphavbeta3 contained several contaminating proteins that were not present in preparations of recombinant alphavbeta3 purified from the transfected CHO cells.
Marcinkiewicz
C
C
The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania, 19140, USA.
Rosenthal
L A
LA
Marcinkiewicz
M M
MM
Kowalska
M A
MA
Niewiarowski
S
S
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D016466:CHO Cells; D002846:Chromatography, Affinity; D006224:Cricetinae; D019483:Disintegrins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005109:Extracellular Matrix / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D016023:Integrins / Q000302:isolation & purification / Q000378:metabolism; D019030:Receptors, Vitronectin / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification*; D014162:Transfection / Q000235:genetics; D019096:Vitronectin / Q000378:metabolism; D014841:von Willebrand Factor / Q000378:metabolism
D000911:Antibodies, Monoclonal; D019483:Disintegrins; D016023:Integrins; D019030:Receptors, Vitronectin; D011994:Recombinant Proteins; D019096:Vitronectin; D014841:von Willebrand Factor