Purification and structure of the intact Ada regulatory protein of Escherichia coli K12, O6-methylguanine-DNA methyltransferase

Y. Nakabeppu, H. Kondo, S. I. Kawabata, S. Iwanaga, M. Sekiguchi

研究成果: ジャーナルへの寄稿記事

104 引用 (Scopus)

抄録

The ada gene of Escherichia coli K12, the regulatory gene for the adaptive response of bacteria to alkylating agents, was cloned and placed under the control of the lac regulatory region on a multicopy runaway plasmid, thereby yielding a hybrid plasmid pYN3059. Ada protein with a molecular weight of about 38,000 was overproduced when cells harboring pYN3059 were incubated at 42°C in the presence of a lac inducer, isopropyl-β-D-thiogalactoside. Taking advantage of overproduction of Ada protein, we purified the protein to apparent physical homogeneity. The purified 38,000-dalton Ada protein transferred the methyl group from the O6-methylguanine residue of alkylated DNA to the Ada protein, per se. Although the Ada protein was degraded to smaller polypeptides when crude extracts or partially purified preparations were incubated in a high ionic-strength buffer at neutral pH, the purified Ada protein remained stable under the same conditions, indicating that the Ada protein may not undergo autodegradation. An amino-terminal sequence and total amino acid composition of the purified Ada protein were in accord with nucleotide sequence of the ada gene, determined by the dideoxy method using M13 phage. It was deduced that Ada protein comprises 354 amino acids and its molecular weight is 39,385. The promoter for the ada gene was determined by S1 nuclease mapping.

元の言語英語
ページ(範囲)7281-7288
ページ数8
ジャーナルJournal of Biological Chemistry
260
発行部数12
出版物ステータス出版済み - 8 28 1985

Fingerprint

Escherichia coli K12
Escherichia coli Proteins
Methyltransferases
Purification
DNA
Proteins
Genes
Plasmids
Molecular Weight
Molecular weight
O-(6)-methylguanine
Thiogalactosides
Bacteriophage M13
Homeless Youth
Isopropyl Thiogalactoside
Amino Acids
Bacteriophages
Nucleic Acid Regulatory Sequences
Alkylating Agents
Regulator Genes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

これを引用

Purification and structure of the intact Ada regulatory protein of Escherichia coli K12, O6-methylguanine-DNA methyltransferase. / Nakabeppu, Y.; Kondo, H.; Kawabata, S. I.; Iwanaga, S.; Sekiguchi, M.

:: Journal of Biological Chemistry, 巻 260, 番号 12, 28.08.1985, p. 7281-7288.

研究成果: ジャーナルへの寄稿記事

@article{ddbe65d11a7742a9917faeebd4fc6f7c,
title = "Purification and structure of the intact Ada regulatory protein of Escherichia coli K12, O6-methylguanine-DNA methyltransferase",
abstract = "The ada gene of Escherichia coli K12, the regulatory gene for the adaptive response of bacteria to alkylating agents, was cloned and placed under the control of the lac regulatory region on a multicopy runaway plasmid, thereby yielding a hybrid plasmid pYN3059. Ada protein with a molecular weight of about 38,000 was overproduced when cells harboring pYN3059 were incubated at 42°C in the presence of a lac inducer, isopropyl-β-D-thiogalactoside. Taking advantage of overproduction of Ada protein, we purified the protein to apparent physical homogeneity. The purified 38,000-dalton Ada protein transferred the methyl group from the O6-methylguanine residue of alkylated DNA to the Ada protein, per se. Although the Ada protein was degraded to smaller polypeptides when crude extracts or partially purified preparations were incubated in a high ionic-strength buffer at neutral pH, the purified Ada protein remained stable under the same conditions, indicating that the Ada protein may not undergo autodegradation. An amino-terminal sequence and total amino acid composition of the purified Ada protein were in accord with nucleotide sequence of the ada gene, determined by the dideoxy method using M13 phage. It was deduced that Ada protein comprises 354 amino acids and its molecular weight is 39,385. The promoter for the ada gene was determined by S1 nuclease mapping.",
author = "Y. Nakabeppu and H. Kondo and Kawabata, {S. I.} and S. Iwanaga and M. Sekiguchi",
year = "1985",
month = "8",
day = "28",
language = "English",
volume = "260",
pages = "7281--7288",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "12",

}

TY - JOUR

T1 - Purification and structure of the intact Ada regulatory protein of Escherichia coli K12, O6-methylguanine-DNA methyltransferase

AU - Nakabeppu, Y.

AU - Kondo, H.

AU - Kawabata, S. I.

AU - Iwanaga, S.

AU - Sekiguchi, M.

PY - 1985/8/28

Y1 - 1985/8/28

N2 - The ada gene of Escherichia coli K12, the regulatory gene for the adaptive response of bacteria to alkylating agents, was cloned and placed under the control of the lac regulatory region on a multicopy runaway plasmid, thereby yielding a hybrid plasmid pYN3059. Ada protein with a molecular weight of about 38,000 was overproduced when cells harboring pYN3059 were incubated at 42°C in the presence of a lac inducer, isopropyl-β-D-thiogalactoside. Taking advantage of overproduction of Ada protein, we purified the protein to apparent physical homogeneity. The purified 38,000-dalton Ada protein transferred the methyl group from the O6-methylguanine residue of alkylated DNA to the Ada protein, per se. Although the Ada protein was degraded to smaller polypeptides when crude extracts or partially purified preparations were incubated in a high ionic-strength buffer at neutral pH, the purified Ada protein remained stable under the same conditions, indicating that the Ada protein may not undergo autodegradation. An amino-terminal sequence and total amino acid composition of the purified Ada protein were in accord with nucleotide sequence of the ada gene, determined by the dideoxy method using M13 phage. It was deduced that Ada protein comprises 354 amino acids and its molecular weight is 39,385. The promoter for the ada gene was determined by S1 nuclease mapping.

AB - The ada gene of Escherichia coli K12, the regulatory gene for the adaptive response of bacteria to alkylating agents, was cloned and placed under the control of the lac regulatory region on a multicopy runaway plasmid, thereby yielding a hybrid plasmid pYN3059. Ada protein with a molecular weight of about 38,000 was overproduced when cells harboring pYN3059 were incubated at 42°C in the presence of a lac inducer, isopropyl-β-D-thiogalactoside. Taking advantage of overproduction of Ada protein, we purified the protein to apparent physical homogeneity. The purified 38,000-dalton Ada protein transferred the methyl group from the O6-methylguanine residue of alkylated DNA to the Ada protein, per se. Although the Ada protein was degraded to smaller polypeptides when crude extracts or partially purified preparations were incubated in a high ionic-strength buffer at neutral pH, the purified Ada protein remained stable under the same conditions, indicating that the Ada protein may not undergo autodegradation. An amino-terminal sequence and total amino acid composition of the purified Ada protein were in accord with nucleotide sequence of the ada gene, determined by the dideoxy method using M13 phage. It was deduced that Ada protein comprises 354 amino acids and its molecular weight is 39,385. The promoter for the ada gene was determined by S1 nuclease mapping.

UR - http://www.scopus.com/inward/record.url?scp=0021879353&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0021879353&partnerID=8YFLogxK

M3 - Article

C2 - 2987251

AN - SCOPUS:0021879353

VL - 260

SP - 7281

EP - 7288

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 12

ER -