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 Eco RI 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 M r 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 β- d -thiogalactopyranoside (IPTG).