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

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

doi:10.1016/j.pep.2006.01.002

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 journal › Article › peer-review

31 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 language	English
Pages (from-to)	607-613
Number of pages	7
Journal	Protein Expression and Purification
Volume	47
Issue number	2
DOIs	https://doi.org/10.1016/j.pep.2006.01.002
Publication status	Published - Jun 2006

Keywords

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

ASJC Scopus subject areas

Biotechnology

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10.1016/j.pep.2006.01.002

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

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.",

keywords = "Cysteine synthase, Nonproteinaceous amino acid, O-Acetylserine sulfhydrylase-B, Recombinant Escherichia coli",

author = "Chunhui Zhao and Yoichi Kumada and Hiroyuki Imanaka and Koreyoshi Imamura and Kazuhiro Nakanishi",

year = "2006",

month = jun,

doi = "10.1016/j.pep.2006.01.002",

language = "English",

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pages = "607--613",

journal = "Protein Expression and Purification",

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T1 - Cloning, overexpression, purification, and characterization of O-acetylserine sulfhydrylase-B from Escherichia coli

AU - Zhao, Chunhui

AU - Kumada, Yoichi

AU - Imanaka, Hiroyuki

AU - Imamura, Koreyoshi

AU - Nakanishi, Kazuhiro

PY - 2006/6

Y1 - 2006/6

N2 - 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.

AB - 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.

KW - Cysteine synthase

KW - Nonproteinaceous amino acid

KW - O-Acetylserine sulfhydrylase-B

KW - Recombinant Escherichia coli

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JO - Protein Expression and Purification

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ER -

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

Abstract

Keywords

ASJC Scopus subject areas

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