Expression of human .alpha.2-macroglobulin cDNA in baby hamster kidney fibroblasts: secretion of high levels of active .alpha.2-macroglobulin

Abstract
Human .alpha.2-macroglobulin (.alpha.2M) 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, .alpha.2M entraps the proteinase molecule in a reaction that involves large conformational changes in .alpha.2M. We describe the molecular cloning of .alpha.2M 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 .alpha.2M with a specific enzyme-linked immunosorbent assay. Human recombinant .alpha.2M (r.alpha.2M), secreted and purified from isolated transfected BHK cell lines, was structurally and functionally compared to .alpha.2M purified from human serum. The results show that r.alpha.2M was secreted from the BHK cells as an active proteinase-binding tetramer with functional thiol esters. Cleavage reactions for .alpha.2M 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, r.alpha.2M-trypsin complex bound to purified human placental .alpha.2M receptor with an affinity indistinguishable from that of a complex formed from serum-derived .alpha.2M and trypsin.