Mini-TnhlyAs: a new tool for the construction of secreted fusion proteins |
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Authors: | I. Gentschev G. Maier A. Kranig W. Goebel |
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Affiliation: | (1) Lehrstuhl für Mikrobiologie, Theodor-Boveri-Institut für Biowissenschaften, Am Hubland, D-97074 Würzburg, Germany;(2) Present address: MPI für Biochemie, D-85152 Martinsried, Germany |
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Abstract: | A simple and efficient procedure for the construction of secreted fusion proteins inEscherichia coli is described that uses a new minitransposon, termed TnhlyAs, carrying the secretion signal (HlyAs) ofE. coli hemolysin (HlyA). This transposon permits the generation of random gene fusions encoding proteins that carry the HlyAs at their C-termini. For the construction of model gene fusions we usedlacZ, encoding the cytoplasmic-galactosidase (-Gal), andphoA, encoding the periplasmic alkaline phosphatase, as target genes. Our data suggest that all-Gal-HlyAs fusion proteins generated are secreted, albeit with varying efficiencies, by the HlyB/HlyD/TolC hemolysin secretion machinery under Sec-proficient conditions. In contrast, the PhoA-HlyAs fusion proteins are efficiently secreted in asecA mutant strain only under SecA-deficient conditions. |
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Keywords: | Escherichia coli hemolysin Minitransposon Secretion of HlyAs-fusion proteins TolC protein |
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