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 translational fusions with the phoA gene (encoding the periplasmic alkaline phosphatase, PhoA) in the three reading frames. One plasmid series carries a multiple cloning site (MCS) followed by a promoterless and leaderless 5'-truncated phoA ('phoA), which in turn is followed by a kanamycin-resistance-encoding gene (kan). Another plasmid series contains two identical inverted MCS flanking the phoA-kan cluster. These latter vectors are devised as phoA-kan cassette delivery vectors. In-frame cloning results in the production of hybrid PhoA proteins which display PhoA 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 promoter and identify the hybrid gene products.
Rodríguez-Quiñones
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Departamento de I+D, Munditest S.A., Palma de Mallorca, Spain.
Hernández-Allés
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Albertí
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Escribá
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Benedí
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VJ
D000469:Alkaline Phosphatase / Q000096:biosynthesis*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005822:Genetic Vectors; D007613:Kanamycin Resistance / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010957:Plasmids; D016133:Polymerase Chain Reaction; D018272:Porins / Q000096:biosynthesis*; D014176:Protein Biosynthesis; D055503:Protein Multimerization; D016364:Reading Frames; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D015183:Restriction Mapping
D017931:DNA Primers; D029968:Escherichia coli Proteins; D018272:Porins; D011993:Recombinant Fusion Proteins; C082922:PhoE protein, E coli; D000469:Alkaline Phosphatase