Molecular cloning of the γ-glutamyltranspeptidase gene from a Pseudomonas strain

Masayuki Ishiye, Mitsuo Yamashita, Mineo Niwa

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

γ-Glutamyltranspeptidase (GGT) was purified from a Pseudomonas sp. strain A14. The purified enzyme was found to be composed of two nonidentical subunits with molecular weights of 39 000 and 22 000 and had a pI of >8.6. The partial N-terminal amino acid sequences of both subunits and some proteolytic fragments were determined. Using mixed oligonucleotides designed from the partial amino acid sequences as hybridization probes, one cosmid clone which contained the GGT gene was isolated from a Pseudomonas sp. strain A14 cosmid genome library, and the DNA sequence of the GGT gene was determined. The nucleotide sequence and the protein sequence analysis revealed that GGT was synthesized as a precursor protein of 575 amino acids and then processed to mature enzyme, presumably after removal of a signal peptide. Comparison of the predicted amino acid sequence of Pseudomonas GGT with published results for Escherichia coli K-12 and rat kidney GGTs shows that the protein sequence of Pseudomonas GGT is 51% and 33% identical to the E. coli and rat GGT sequences, respectively. Higher similarity is observed among the small subunits, which have been thought to have a binding site for the γ-glutamyl residue. Expression of the cloned Pseudomonas GGT gene in E. coli was subjected to Western blot analysis using antibody raised against the purified GGT. This suggested that processing of the precursor protein to its subunits is temperature-dependent, because the amount of mature GGT protein was increased when the culture was performed at low temperature.

Original languageEnglish
Pages (from-to)323-331
Number of pages9
JournalBiotechnology Progress
Volume9
Issue number3
Publication statusPublished - 1993
Externally publishedYes

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Pseudomonas
molecular cloning
amino acid sequences
genes
Escherichia coli K12
Escherichia coli
nucleotide sequences
proteins
rats
enzymes
oligonucleotides
signal peptide
binding sites
temperature
Western blotting
sequence analysis
kidneys
clones
molecular weight
amino acids

ASJC Scopus subject areas

  • Food Science
  • Biotechnology
  • Microbiology

Cite this

Molecular cloning of the γ-glutamyltranspeptidase gene from a Pseudomonas strain. / Ishiye, Masayuki; Yamashita, Mitsuo; Niwa, Mineo.

In: Biotechnology Progress, Vol. 9, No. 3, 1993, p. 323-331.

Research output: Contribution to journalArticle

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