Expression and Purification of Correctly Processed, Active Human TACE Catalytic Domain inSaccharomyces cerevisiae

Abstract Human tumor necrosis factor-α (TNFα) converting enzyme (TACE) releases soluble TNFα from cells. It is a member of the adamalysin family of metalloproteases. A truncated form of TACE cDNA was expressed in Saccharomyces cerevisiae and purified to homogeneity in order to study TACE structure and function. Recombinant TACE was expressed as a preproprotein including the pro- and catalytic (PROCAT) domains fused to the yeast α-factor leader. A C-terminal immunoreactive FLAG peptide was added for Western blot detection and anti-FLAG antibody column purification. We constructed two glycosylation mutant PROCAT TACE isoforms to facilitate purification. A PROCAT isoform, mutated to eliminate two N-linked glycosylation sites, was buffer exchanged and purified to homogeneity by ion exchange chromatography and an anti-FLAG antibody affinity step. N-terminal sequence analysis showed that the mutant preproprotein was processed in yeast at the furin protease cleavage site and yielded an active catalytic domain which has TNFα peptide-specific protease activity. Mass spectrometry of the purified catalytic domain showed that removal of both N-linked sites results in a homogeneous sized polypeptide lacking further posttranslational modifications.