A NOVEL PLASMID SERIES FOR IN-VITRO PRODUCTION OF PHOA TRANSLATIONAL FUSIONS AND ITS USE IN THE CONSTRUCTION OF ESCHERICHIA-COLI PHOE-PHOA HYBRID PROTEINS

We have developed a series of vectors for easy construction of transla tional fusions with the phoA gene (encoding the periplasmic alkaline p hosphatase, PhoA) in the three reading frames. One plasmid series carr ies a multiple cloning site (MCS) followed by a promoterless and leade rless 5'-truncated phoA ('phoA), which in turn is followed by a kanamy cin-resistance-encoding gene (kan). Another plasmid series contains tw o identical inverted MCS flanking the phoA-kan cluster. These latter v ectors are devised as phoA-kan cassette delivery vectors. In-frame clo ning results in the production of hybrid PhoA proteins which display P hoA activity if successfully exported beyond the cytoplasmic membrane. In order to test these vectors, we have constructed hybrid PhoE::PhoA proteins, which were used to analyze the activity of the phoE promote r and identify the hybrid gene products.