ONE-STEP AFFINITY PURIFICATION OF RECOMBINANT ALPHA(V)BETA(3) INTEGRIN FROM TRANSFECTED CELLS

Vitronectin receptor (alpha(v) beta(3) integrin) is present on the sur face of many types of cells. We describe a simple, fast, and reliable method of purification of recombinant human alpha(v) beta(3) from Chin ese hamster ovary (CHO) cells transfected with alpha(v) beta(3) (VNRC3 cells). The method consists of two steps: lysis of the cells and affi nity chromatography of the lysate on a GRGDSPK-Sepharose column. The y ield of the procedure was about 79%. The purified receptor migrated as two bands on a silver stained SDS-polyacrylamide gel, corresponding t o the alpha and beta(3) subunits, and was recognized by monoclonal ant ibodies directed against alpha(v) and the alpha(v) beta(3) complex, bu t not by monoclonal antibody specific for the alpha(IIb)beta(3) comple x. This receptor also bound to immobilized vitronectin, von Willebrand factor, and echistatin. However, binding to immobilized fibrinogen wa s not observed. Purified recombinant alpha(v) beta(3) demonstrated gre ater immunoreactivity with LM 609, an alpha(v) beta(3) complex-specifi c monoclonal antibody, than alpha(v) beta(3) purified from placenta. A s visualized by SDS-polyacrylamide gel electrophoresis, preparations o f placenta-derived alpha(v) beta(3) contained several contaminating pr oteins that were not present in preparations of recombinant alpha(v) b eta(3) purified from the transfected CHO cells. (C) 1996 Academic Pres s, Inc.