CHINESE-HAMSTER RHODANESE CDNA - ACTIVITY OF THE EXPRESSED PROTEIN ISNOT BLOCKED BY A C-TERMINAL EXTENSION

We describe a cDNA from Chinese hamster ovary cells which encodes a pr otein 91 and 96% identical to bovine and rat mitochondrial rhodaneses, respectively. Recombinant protein was expressed from the cDNA in Esch erichia coil, purified to homogeneity, and found to have kinetic prope rties nearly indistinguishable from those of the bovine enzyme, the on ly cloned rhodanese previously verified by characterization of the rec ombinant protein, The carboxyl-terminus of the enzyme is characterized by a duplicated tripeptide, which can be proteolytically processed in vivo. We constructed a mutant in which the last 5 amino acids were re placed by 28 residues of unrelated sequence. This protein was expresse d, purified, and found to have kinetic constants similar to those of t he wild-type enzyme. The functionally verified Chinese hamster rhodane se cDNA encodes a protein of 297 residues and differs from the rat enz yme at 13 positions. None of these substitutions occurs at residues su ggested to play essential roles in catalysis or structural stabilizati on. (C) 1995 Academic Press, Inc.