Plasmid mutagenesis by PCR for high-level expression of para-hydroxybenzoate hydroxylase

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.