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Expression and purification of an immunogenic SUMO-OmpC fusion protein of Salmonella Typhimurium in Escherichia coli.
Biologicals ( IF 1.7 ) Pub Date : 2019-10-25 , DOI: 10.1016/j.biologicals.2019.10.010
Prejit 1 , Prakasam Thanka Pratheesh 2 , Soman Nimisha 2 , Vergis Jess 1 , Karthikeyan Asha 2 , Rajesh Kumar Agarwal 3
Affiliation  

Salmonella is found to be a major causes of food borne diseases globally. Poultry products contaminated with this pathogen is one of the major sources of infections in humans. Outer membrane protein C (OmpC) of Salmonella Typhimurium is a promising DNA vaccine candidate to mitigate Salmonella infection in poultry. However, the large-scale production of bioactive recombinant OmpC (rOmpC) protein is hindered due to the formation of inclusion bodies in Escherichia coli. The objective of this work was to attain high level expression of rOmpC protein, purify and evaluate its functional properties. The ompC gene was optimized and fused with small ubiquitin-related modifier (SUMO) gene for high level expression as soluble protein. The fusion protein with ~58 kDa molecular weight was observed on SDS-PAGE gel. The expression levels of rOmpC fusion protein reached maximum of 38% of total soluble protein (TSP) after 8 h of 0.2% rhamnose induction. Protein purification was carried out using nickel nitrilotriacetic acid (Ni-NTA) purification column. Western blot were performed to analyse expression and immunoreactivity of rOmpC fusion protein. The results indicate that SUMO fusion system is ideal for large scale production of functional rOmpC fusion protein expression in E. coli.



中文翻译:

鼠伤寒沙门氏菌免疫原性SUMO-OmpC融合蛋白在大肠杆菌中的表达和纯化。

沙门氏菌是全球食源性疾病的主要原因。被这种病原体污染的家禽产品是人类感染的主要来源之一。鼠伤寒沙门氏菌的外膜蛋白C(OmpC)是缓解禽类沙门氏菌感染的有前途的DNA疫苗候选物。然而,由于在大肠杆菌中形成包涵体,阻碍了生物活性重组OmpC(rOmpC)蛋白的大规模生产。这项工作的目的是获得高表达的rOmpC蛋白,纯化和评估其功能特性。在OMPC基因经过优化,并与小的泛素相关修饰子(SUMO)基因融合,可作为可溶性蛋白高水平表达。在SDS-PAGE凝胶上观察到分子量约为58 kDa的融合蛋白。在0.2%鼠李糖诱导8小时后,rOmpC融合蛋白的表达水平最高达到了总可溶性蛋白(TSP)的38%。使用次氮基三乙酸镍(Ni-NTA)纯化柱进行蛋白质纯化。进行了蛋白质印迹分析rOmpC融合蛋白的表达和免疫反应性。结果表明,SUMO融合系统是在大肠杆菌中大规模生产功能性rOmpC融合蛋白表达的理想选择。

更新日期:2019-10-25
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