EXPRESSION OF HUMAN ALPHA-2-MACROGLOBULIN CDNA IN BABY HAMSTER-KIDNEY FIBROBLASTS - SECRETION OF HIGH-LEVELS OF ACTIVE ALPHA-2-MACROGLOBULIN

Human α2-macroglobulin (α2M) is a unique 720-kDa proteinase inhibitor with a broad specificity. Unlike most other proteinase inhibitors, it does not inhibit proteolytic activity by blocking the active site of the proteinase. During complex formation with a proteinase, α2M entraps the proteinase molecule in a reaction that involves large conformational changes in α2M. We describe the molecular cloning of α2M cDNA from the human hepatoblastoma cell line HepG2. The cDNA was subcloned under control of the adenovirus major late promoter in a mammalian expression vector and introduced into the baby hamster kidney (BHK) cell line. Transformed clones were isolated and tested for production of human α2M with a specific enzyme-linked immunosorbent assay. Human recombinant α2M (rα2M), secreted and purified from isolated transfected BHK cell lines, was structurally and functionally compared to α2M purified from human serum. The results show that rα2M was secreted from the BHK cells as an active proteinase-binding tetramer with functional thiol esters. Cleavage reactions of rα2M with methylamine and trypsin showed that the recombinant product, which was correctly processed at the N-terminus, exhibited molecular characteristics similar to those of the human serum derived reference. Moreover, rα2M-trypsin complex bound to purified human placental α2M receptor with an affinity indistinguishable from that of a complex formed from serum-derived α2M and trypsin. © 1990, American Chemical Society. All rights reserved.