Chinese Hamster Rhodanese cDNA

Activity of the Expressed Protein Is Not Blocked by a C-Terminal Extension

R. J. Trevino, J. Hunt, P. M. Horowitz, John Chirgwin

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)693-699
Number of pages7
JournalProtein Expression and Purification
Volume6
Issue number5
DOIs
StatePublished - Oct 1995
Externally publishedYes

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Thiosulfate Sulfurtransferase
Cricetulus
Complementary DNA
Enzymes
Recombinant Proteins
Rats
Proteins
Kinetics
Catalysis
Escherichia coli
Ovary
Substitution reactions
Stabilization
Cells
Amino Acids

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry

Cite this

Chinese Hamster Rhodanese cDNA : Activity of the Expressed Protein Is Not Blocked by a C-Terminal Extension. / Trevino, R. J.; Hunt, J.; Horowitz, P. M.; Chirgwin, John.

In: Protein Expression and Purification, Vol. 6, No. 5, 10.1995, p. 693-699.

Research output: Contribution to journalArticle

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