Cloning and expression of the major porin protein gene opcP of Burkholderia (formerly Pseudomonas) cepacia in Escherichia coli.
The outer membrane protein OpcP1 of Burkholderia (formerly Pseudomonas) cepacia is one of the subunits forming the porin oligomer OpcPO by non-covalent association. OpcP1 was cleaved with lysyl endopeptidase and the N-terminal amino acid (aa) sequences of the polypeptide fragments were determined. Based on the sequence information, we cloned the opcP gene on a 10-kb EcoRI DNA fragment of the B. cepacia ATCC25416 chromosome. Nucleotide (nt) sequencing revealed a 1086-bp open reading frame (ORF), encoding a 361-aa polypeptide with a signal sequence of 20 residues. The predicted opcP gene encoded a mature protein of Mr 35,696, which agrees well with the value observed previously on SDS-PAGE. The opcP was sub-cloned into pTrc99A and introduced into Escherichia coli. Immunoblot analysis using murine antiserum specific to OpcP1 visualized the protein expressed in the E. coli cells after induction by isopropyl beta-D-thiogalactopyranoside (IPTG).
Tsujimoto
H
H
Department of Microbiology, Kyoto Pharmaceutical University, Yamashina, Japan.
Gotoh
N
N
Yamagishi
J
J
Oyamada
Y
Y
Nishino
T
T
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics*; D001483:Base Sequence; D016956:Burkholderia cepacia / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D001425:Bacterial Outer Membrane Proteins; D011994:Recombinant Proteins; C074517:opc protein, bacteria