Cloning and overexpression of Bacillus cereus penicillin-binding protein 3 gene in Escherichia coli.
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 x 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.
Miyamoto
Takahisa
T
Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka, Japan. tmiyamot@agr.kyushu-u.ac.jp
Sayed
Md Abu
MA
Sasahara
Ryo
R
Sukimoto
Kouji
K
Umezaki
Akiko
A
Honjoh
Ken-ichi
K
Iio
Masayaoshi
M
Hatano
Shoji
S
D000595:Amino Acid Sequence; D001409:Bacillus cereus; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli; D015870:Gene Expression; D008969:Molecular Sequence Data; D002267:Muramoylpentapeptide Carboxypeptidase / Q000235:genetics* / Q000302:isolation & purification; D010455:Peptides / Q000032:analysis; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid
D002352:Carrier Proteins; D018076:DNA, Complementary; D010455:Peptides; D002267:Muramoylpentapeptide Carboxypeptidase