Wozniak D J, Han X Y, Galloway D R
Department of Microbiology, Ohio State University, Columbus 43210, USA.
Appl Environ Microbiol. 1995 May;61(5):1739-44. doi: 10.1128/aem.61.5.1739-1744.1995.
To express recombinant forms of Pseudomonas aeruginosa exotoxin A in high yield, we have developed a nontoxigenic strain of P. aeruginosa derived from the hypertoxigenic strain PA103. The nontoxigenic strain, designated PA103A, was produced by the excision marker rescue technique to replace the toxA structural gene in PA103 with an insertionally inactivated toxA gene. The PA103A strain (ToxA-) was used subsequently as the host strain for the expression and production of several recombinant versions of exotoxin A, and the results were compared with exotoxin A production in other P. aeruginosa and Escherichia coli strains. Use of the PA103A strain transformed with the high-copy-number pRO1614 plasmid bearing various toxA alleles resulted in final purification yields of exotoxin A averaging 23 mg/liter of culture. By comparison, exotoxin A production in other expression systems and host strains yields approximately 1/4 to 1/10 as much toxin.
为了高产表达铜绿假单胞菌外毒素A的重组形式，我们构建了一种源自高毒力菌株PA103的无毒力铜绿假单胞菌菌株。通过切除标记拯救技术，用插入失活的toxA基因取代PA103中的toxA结构基因，构建了无毒力菌株PA103A。随后，PA103A菌株（ToxA-）被用作宿主菌株，用于表达和生产几种外毒素A的重组变体，并将结果与其他铜绿假单胞菌和大肠杆菌菌株中外毒素A的产量进行比较。使用携带各种toxA等位基因的高拷贝数pRO1614质粒转化的PA103A菌株，外毒素A的最终纯化产量平均为每升培养物23毫克。相比之下，其他表达系统和宿主菌株中外毒素A的产量约为该毒素产量的1/4至1/10。
