title: Cloning and expression of the major porin protein gene opcP of Burkholderia (formerly Pseudomonas) cepacia in Escherichia coli.
abstract: 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).
authors.affiliation: Department of Microbiology, Kyoto Pharmaceutical University, Yamashina, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Tsujimoto
authors.forename: N
authors.initials: N
authors.lastname: Gotoh
authors.forename: J
authors.initials: J
authors.lastname: Yamagishi
authors.forename: Y
authors.initials: Y
authors.lastname: Oyamada
authors.forename: T
authors.initials: T
authors.lastname: Nishino
mesh_terms: 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
chemical_list: D001425:Bacterial Outer Membrane Proteins; D011994:Recombinant Proteins; C074517:opc protein, bacteria