Conrad B, Savchenko R S, Breves R, Hofemeister J
Institut fur Pflanzengenetik und Kulturpflanzenforschung, Gatersleben, Germany.
Mol Gen Genet. 1996 Feb 5;250(2):230-6. doi: 10.1007/BF02174183.
The adaptation and application of the Escherichia coli T7 RNA polymerase system for regulated and promoter-specific gene expression in Bacillus subtilis is reported. The expression cassette used in Bacillus subtilis was tightly regulated and T7 RnA polymerase (T7 RNAP)appeared 30 minutes after induction. The efficiency of T7 promoter-specific gene expression in B.subtilis was studied using one secretory and two cytosolic proteins of heterologous origin. The accumulation of E. coli beta-galactosidase, as well as a 1,4-beta-glucosidase from Thermoanaerobacter brockii in B. subtilis after T7 RNAP induction was strongly enhanced by rifampicin inhibition of host RNAP activity. The alpha-amylase of Thermactinomyces 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 chi-amylase secretion, but unexpectedly, after a short period, also prevented its further (intra)cellular accumulation.
报道了大肠杆菌T7 RNA聚合酶系统在枯草芽孢杆菌中用于调控和启动子特异性基因表达的适应性及应用。枯草芽孢杆菌中使用的表达盒受到严格调控，诱导后30分钟出现T7 RNA聚合酶（T7 RNAP）。利用一种分泌型和两种异源胞质蛋白研究了枯草芽孢杆菌中T7启动子特异性基因表达的效率。利福平抑制宿主RNAP活性后，T7 RNAP诱导后枯草芽孢杆菌中大肠杆菌β-半乳糖苷酶以及嗜热栖热放线菌的1,4-β-葡萄糖苷酶的积累显著增强。普通嗜热放线菌的分泌蛋白α-淀粉酶在T7 RNAP诱导后10 - 20小时在培养上清液中的积累量高达约70 mg/l，但也沉积在细胞组分中。添加利福平抑制了α-淀粉酶的分泌，但出乎意料的是，短时间后也阻止了其在细胞内（外）的进一步积累。
