Identification of Genes Encoding Secreted Proteins Using Mini-OphoA Mutagenesis

Protein fusions are invaluable tools for the genetic studies involving the mechanisms of protein export in bacteria. In 1985, Hoffman and Wright developed an in vitro fusion approach that allowed for fusions of the gene encoding Escherichia coli alkaline phosphatase to a variety of cloned genes (1 ). The modified phoA gene employed in these studies, designated 'phoA , resulted in the production of a highly active alkaline phosphatase protein missing its signal sequence (1 ). This approach is based on the fact that bacterial alkaline phosphatase is normally periplasmic and must be located extracytoplasmically to be active, i.e., export is essential for high levels of alkaline phosphatase activity (1 ). Through the fusion of 'phoA to portions of heterologous genes containing signal sequences, export from the cytoplasm and subsequent PhoA activity can be observed (1 ).