Chinese hamster rhodanese cDNA: activity of the expressed protein is not blocked by a C-terminal extension.
We describe a cDNA from Chinese hamster ovary cells which encodes a protein 91 and 96% identical to bovine and rat mitochondrial rhodaneses, respectively. Recombinant protein was expressed from the cDNA in Escherichia coli, purified to homogeneity, and found to have kinetic properties nearly indistinguishable from those of the bovine enzyme, the only cloned rhodanese previously verified by characterization of the recombinant 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 replaced by 28 residues of unrelated sequence. This protein was expressed, purified, and found to have kinetic constants similar to those of the wild-type enzyme. The functionally verified Chinese hamster rhodanese cDNA encodes a protein of 297 residues and differs from the rat enzyme at 13 positions. None of these substitutions occurs at residues suggested to play essential roles in catalysis or structural stabilization.
Department of Biochemistry, University of Texas Health Science Center at San Antonio 78284, USA.
R J
RJ
Trevino
J
J
Hunt
P M
PM
Horowitz
J M
JM
Chirgwin
D000818:Animals; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D011994:Recombinant Proteins; D013884:Thiosulfate Sulfurtransferase