Expression of the cell-binding domain of human fibronectin in E. coli. Identification of sequences promoting full to minimal adhesive function |
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Authors: | M Obara M S Kang S Rocher-Dufour A Kornblihtt J P Thiery K M Yamada |
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Affiliation: | 1. Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA;2. Institut d''Embryologie, CNRS et College de France, 94130 Nogent sur Marne Cedex, France;3. Instituto de Investigaciones en Ingenieria Genetica y Biologia Molecular (INGEBI-CONICET), Buenos Aires, Argentina |
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Abstract: | Two cDNA subfragments containing the cell-attachment site of human fibronectin (FN) were expressed as beta-galactosidase fusion proteins in E. coli. The products were purified to homogeneity by monoclonal antibody affinity chromatography and assayed for activity in a standard cell-adhesion assay. A fusion protein containing an 80 kDa fragment of human FN appeared functionally equivalent to intact FN purified from human plasma, whereas a truncated fusion protein of 33 kDa still containing a previously postulated cell-attachment site was approx. 50-fold less active. Our study establishes a system for analyzing adhesive protein function by DNA manipulation, rules out any major role for eukaryotic post-translational modifications in FN adhesive function, and localizes additional functional activity to a 1.3 kb region. |
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