Cloning and overexpression of Bacillus cereus penicillin-binding protein 3 gene in Escherichia coli

Takahisa Miyamoto, Md Abu Sayed, Ryo Sasahara, Kouji Sukimoto, Akiko Umezaki, Ken-ichi Honjoh, Masayaoshi Iio, Shoji Hatano

Research output: Contribution to journalArticlepeer-review

Abstract

The pbp3 gene encoding PBP3 of Bacillus cereus was cloned and sequenced. For this purpose, PBP3 was first purified from B. cereus ts-4, and N-terminal amino acid sequences of the peptides obtained from the protease digests of the protein were analyzed. The B. cereus ts-4 pbp3 gene consisted of an open reading frame of 1,986 bp encoding 662 amino acid residues with a calculated molecular mass of 73,044 Da. The active site-motifs SXXK, SXN, and KTG are present at the positions 393, 452, and 590, respectively, in the deduced amino acid sequence. The pbp3 structural gene was ligated into the pET17×b expression vector and pET-pbp3 was constructed. A protein was produced by the cells of E. coli carrying pET-pbp3. The produced protein migrated at about 75 kDa in SDS-polyacrylamide gel and strongly reacted with biotinylated ampicillin.

Original languageEnglish
Pages (from-to)44-50
Number of pages7
JournalBioscience, Biotechnology and Biochemistry
Volume66
Issue number1
DOIs
Publication statusPublished - Jan 1 2002

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Analytical Chemistry
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Molecular Biology
  • Organic Chemistry

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