枯草芽孢杆菌
T7 RNA聚合酶
生物
核糖体结合位点
质粒
基因
表达式向量
分子生物学
基因表达
转化(遗传学)
生物化学
重组DNA
大肠杆菌
核糖体
核糖核酸
遗传学
细菌
噬菌体
作者
Jing Ye,Yunjie Li,Yan Bai,Ting Zhang,Wei Jiang,Ting Shi,Zijian Wu,Y.‐H. Percival Zhang
标识
DOI:10.1186/s40643-022-00540-4
摘要
Abstract To mimic the Escherichia coli T7 protein expression system, we developed a facile T7 promoter-based protein expression system in an industrial microorganism Bacillus subtilis . This system has two parts: a new B. subtilis strain SCK22 and a plasmid pHT7. To construct strain SCK22, the T7 RNA polymerase gene was inserted into the chromosome, and several genes, such as two major protease genes, a spore generation-related gene, and a fermentation foam generation-related gene, were knocked out to facilitate good expression in high-density cell fermentation. The gene of a target protein can be subcloned into plasmid pHT7, where the gene of the target protein was under tight control of the T7 promoter with a ribosome binding site (RBS) sequence of B. subtilis (i.e., AAGGAGG). A few recombinant proteins (i.e., green fluorescent protein, α-glucan phosphorylase, inositol monophosphatase, phosphoglucomutase, and 4-α-glucanotransferase) were expressed with approximately 25–40% expression levels relative to the cellular total proteins estimated by SDS-PAGE by using B. subtilis SCK22/pHT7-derived plasmid. A fed-batch high-cell density fermentation was conducted in a 5-L fermenter, producing up to 4.78 g/L inositol monophosphatase. This expression system has a few advantageous features, such as, wide applicability for recombinant proteins, high protein expression level, easy genetic operation, high transformation efficiency, good genetic stability, and suitability for high-cell density fermentation. Graphical Abstract
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