title: Plasmid mutagenesis by PCR for high-level expression of para-hydroxybenzoate hydroxylase.
abstract: We report a PCR deletion mutagenesis method for the exact positioning of a foreign gene (pobA) in the lac operon of an expression plasmid in place of the lacZ protein code. This method requires the synthesis of four oligonucleotides and three PCR reactions to delete unwanted bases and retain the nucleotide sequence naturally found between the lac promoter and the protein code. The engineered plasmid results in the production of at least 40% of the cellular protein as the foreign polypeptide. In the example presented the expression of the protein is high even with a substantial difference in codon usage between the host (Escherichia coli) and a foreign gene from Pseudomonas aeruginosa. Some of the polypeptide produced has the ame properties as native protein and is easily purified. The remainder is present as insoluble inclusion bodies. This method of plasmid refinement may be applicable to the expression of many proteins.
authors.affiliation: Department of Biochemistry, Microbiology and Nutrition, University of New England, Armidale, NSW, Australia.
authors.forename: G R
authors.initials: GR
authors.lastname: Moran
authors.forename: B
authors.initials: B
authors.lastname: Entsch
mesh_terms: D015109:4-Hydroxybenzoate-3-Monooxygenase / Q000096:biosynthesis* / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics; D001483:Base Sequence; D003062:Codon; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D005822:Genetic Vectors; D007763:Lac Operon; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D011550:Pseudomonas aeruginosa / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D017384:Sequence Deletion
chemical_list: D001426:Bacterial Proteins; D003062:Codon; D011993:Recombinant Fusion Proteins; D015109:4-Hydroxybenzoate-3-Monooxygenase