Efficient expression system of human recombinant laminin-5.
Laminin-5, a heterotrimer of laminin alpha3, beta3, and gamma2 chains, is an essential component of various epithelial basement membranes, and it strongly promotes cellular adhesion and motility in vitro. In this study, we established an efficient expression system of human recombinant laminin-5 (rLN5), in which full-length cDNAs encoding the human laminin alpha3, beta3, and gamma2 chains were introduced into the human embryonic kidney cell line HEK293. rLN5 was purified from the conditioned medium of the HEK293 transfectant (LN5-HEK) by immuno-affinity chromatography in a yield of 1 mg protein/liter, about 10 times higher than that of a natural LN5 from human gastric cancer cells. rLN5 was indistinguishable from the natural LN5 in its protein composition and biological activity. In addition, analysis of HEK293 transfectants expressing two exogenous LN5 subunits showed that the alpha3/gamma2 chains and the beta3/gamma2 chains, but not the alpha3/beta3 chains, were secreted as heterodimers, suggesting an important role of the gamma2 chain in the association of the three LN5 subunits. The expression system of rLN5 can be used as an important tool to understand the biological functions of this laminin and may be applicable to future regenerative medicine.
D000818:Animals; D002448:Cell Adhesion / Q000502:physiology; D015815:Cell Adhesion Molecules / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002465:Cell Movement / Q000502:physiology; D002478:Cells, Cultured; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D015151:Immunoblotting; D007668:Kidney / Q000378:metabolism; D007797:Laminin / Q000235:genetics / Q000378:metabolism; D008858:Microscopy, Phase-Contrast; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured; D000097794:Kalinin
D015815:Cell Adhesion Molecules; D017931:DNA Primers; D018076:DNA, Complementary; D007797:Laminin; D011994:Recombinant Proteins; D000097794:Kalinin