Cloning, overexpression, purification, and characterization of O-acetylserine sulfhydrylase-B from Escherichia coli

Chunhui Zhao, Yoichi Kumada, Hiroyuki Imanaka, Koreyoshi Imamura, Kazuhiro Nakanishi

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

O-Acetylserine sulfhydrylase-B (OASS-B, EC 2.5.1.47) is one of the two isozymes produced by Escherichia coli that catalyze the synthesis of l-cysteine from O-acetyl-l-serine and sulfide. The cysM gene encoding OASS-B was cloned and the enzyme was overexpressed in E. coli using pUC19 with a lacUV5 promoter. The enzyme was purified to homogeneity, as evidenced by SDS-PAGE. Approximately 300 mg of purified OASS-B was obtained from 1600 mL of culture broth with a purification yield of 60% or higher. The purified OASS-B was characterized and its properties compared with OASS-A. OASS-B did not form a complex with E. coli serine acetyltransferase (SAT, EC 2.3.1.30) and showed a wide range of substrate specificity in nonproteinaceous amino acid synthesis.

Original languageEnglish
Pages (from-to)607-613
Number of pages7
JournalProtein Expression and Purification
Volume47
Issue number2
DOIs
Publication statusPublished - Jun 2006

Fingerprint

Cysteine Synthase
Cloning
Escherichia coli
Purification
Organism Cloning
Serine O-Acetyltransferase
Gene encoding
Sulfides
Enzymes
Substrate Specificity
Serine
Isoenzymes
Cysteine
Polyacrylamide Gel Electrophoresis
Amino Acids
Substrates
Genes

Keywords

  • Cysteine synthase
  • Nonproteinaceous amino acid
  • O-Acetylserine sulfhydrylase-B
  • Recombinant Escherichia coli

ASJC Scopus subject areas

  • Biochemistry

Cite this

Cloning, overexpression, purification, and characterization of O-acetylserine sulfhydrylase-B from Escherichia coli. / Zhao, Chunhui; Kumada, Yoichi; Imanaka, Hiroyuki; Imamura, Koreyoshi; Nakanishi, Kazuhiro.

In: Protein Expression and Purification, Vol. 47, No. 2, 06.2006, p. 607-613.

Research output: Contribution to journalArticle

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AU - Kumada, Yoichi

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AU - Imamura, Koreyoshi

AU - Nakanishi, Kazuhiro

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