A T7 promoter-specific, inducible protein expression system forBacillus subtilis |
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| Authors: | Birgit Conrad Regina S Savchenko Roland Breves and Jürgen Hofemeister |
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| Institution: | (1) Institut für Pflanzengenetik und Kulturpflanzenforschung, Corrensstrasse 03, D-06466 Gatersleben, Germany;(2) Present address: University of North Texas, Health Science Center at Fort Worth, 3500 Camp Bowie Boulevard, 76107 Fort Worth, TX, USA |
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| Abstract: | The adaptation and application of theEscherichia coli T7 RNA polymerase system for regulated and promoter-specific gene expression inBacillus subtilis is reported. The expression cassette used inBacillus subtilis was tightly regulated and T7 RNA polymerase (T7 RNAP) appeared 30 min after induction. The efficiency of T7 promoter-specific gene expression inB. subtilis was studied using one secretory and two cytosolic proteins of heterologous origin. The accumulation ofE. coli  -galactosidase, as well as a 1,4--glucosidase fromThermoanaerobacter brockii inB. subtilis after T7 RNAP induction was strongly enhanced by rifampicin inhibition of host RNAP activity. The -amylase ofThermoactinomyces vulgaris, a secretory protein, was found to accumulate in the culture supernatant up to levels of about 70 mg/l 10–20 h after T7 RNAP induction, but was also deposited in cellular fractions. The addition of rifampicin inhibited-amylase secretion, but unexpectedly, after a short period, also prevented its further (intra)cellular accumulation |
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| Keywords: | Bacillus subtilis T7 RNA polymerase Promoter specificity Gene expression Protein secretion |
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