Ribas A V, Ho P L, Tanizaki M M, Raw I, Nascimento A L
Center of Biotechnology, Instituto Butantan, Av. Vital Brasil, 1500, CEP 05503-900, São Paulo, SP, Brazil.
Biotechnol Appl Biochem. 2000 Apr;31(2):91-4. doi: 10.1042/ba19990084.
An insert of Clostridium tetani DNA corresponding to fragment C of tetanus toxin was amplified by PCR. This 1.4 kb fragment was cloned into the high-expression vector pET32a, under control of the T7 promoter. Expression of this plasmid in Escherichia coli BL21(DE3) resulted in the production of a fusion protein ( approximately 62 kDa) consisting of 112 amino acids of thioredoxin and approximately 450 amino acids of fragment C. This fusion protein was recognized by anti-tetanus toxoid antiserum in an ELISA and on immunoblots. The recombinant fragment-C-thioredoxin protein was purified significantly in one step by Ni(2+)-chelate Sepharose, the final yield being approximately 35 mg/l. Immunization of animals with the recombinant protein produced antibodies that were able to recognize the tetanus toxin. By using this gene-fusion expression system we produced soluble fragment C of tetanus toxin in a high yield, preventing many problems inherent in the use of other expression systems that produce either insoluble fragment C in inclusion bodies, or a soluble form, but in low yield, using E. coli as the expression host.
通过PCR扩增出与破伤风毒素片段C相对应的破伤风梭菌DNA片段。将这个1.4 kb的片段克隆到受T7启动子控制的高表达载体pET32a中。该质粒在大肠杆菌BL21(DE3)中的表达产生了一种融合蛋白（约62 kDa），它由112个氨基酸的硫氧还蛋白和约450个氨基酸的片段C组成。在ELISA和免疫印迹中，这种融合蛋白能被抗破伤风类毒素抗血清识别。重组的片段C-硫氧还蛋白通过Ni(2+) - 螯合琼脂糖一步法得到了显著纯化，最终产量约为35 mg/l。用重组蛋白免疫动物产生了能够识别破伤风毒素的抗体。通过使用这种基因融合表达系统，我们以高产率生产出了可溶性的破伤风毒素片段C，避免了使用其他表达系统时固有的许多问题，这些问题包括在包涵体中产生不溶性片段C，或者产生可溶性形式但产量低，而这里是以大肠杆菌作为表达宿主。
