Expression, production, and characterization of full-length vitronectin in Escherichia coli.
Vitronectin (VN) is one of the primary adhesive proteins in serum and serves to promote the attachment and spreading of a wide variety of cell types to tissue culture plastic. In this study, the pGEX2t expression vector was used to express full-length human VN as a GST-tagged fusion protein in Escherichia coli. GST/VN production was induced with IPTG and the protein was found to localize to inclusion bodies. The inclusion bodies were isolated from cell lysates, washed once with 2 M urea and Triton X-100, and then solubilized with 8 M urea in the presence of a reducing compound. Solubilized GST/VN was purified by heparin affinity chromatography and refolded by dialysis against phosphate buffered saline. Approximately 40 mg of GST/VN was recovered from 1L of bacterial culture. Purified GST/VN migrated at the predicted molecular mass on SDS-PAGE and was recognized by both anti-GST and anti-VN antibodies. GST/VN bound to heparin and promoted cell adhesion, spreading, and growth to a similar extent as that observed with plasma-derived VN. As such, the production of recombinant VN in bacteria represents a rapid and convenient method to produce large quantities of VN for cellular studies.
Wojciechowski
Katherine
K
Department of Pharmacology and Physiology, University of Rochester Medical Center, 601 Elmwood Ave., Box 711, NY 14642, USA.
Chang
Cecilia H
CH
Hocking
Denise C
DC
D000818:Animals; D001667:Binding, Competitive; D002448:Cell Adhesion / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics; D006493:Heparin / Q000737:chemistry; D006801:Humans; D039564:Integrin alphaV / Q000494:pharmacology; D039661:Integrin beta3 / Q000494:pharmacology; D051379:Mice; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000494:pharmacology; D019096:Vitronectin / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology*
D039564:Integrin alphaV; D039661:Integrin beta3; D011993:Recombinant Fusion Proteins; D019096:Vitronectin; D006493:Heparin; D005982:Glutathione Transferase