Chemical Engineering Department, Federal University of São Carlos, Rodovia Washington Luís, km 235, CP 676, São Carlos, SP CEP 13565-905, Brazil.
Curr Microbiol. 2012 Oct;65(4):369-74. doi: 10.1007/s00284-012-0171-y. Epub 2012 Jun 24.
This work reports the cloning, expression, and purification of a 42-kDa fragment of the SpaA protein from Erysipelothrix rhusiopathiae, the main antigenic candidate for a subunit vaccine against swine erysipelas. The use of an auto-induction protocol to improve heterologous protein expression in recombinant Escherichia coli cultures was also investigated. The cellular growth pattern and metabolite formation were evaluated under different induction conditions. The His-tagged protein was over-expressed as inclusion bodies, and was purified by a single chromatography step under denaturing conditions. Auto-induction conditions were shown to be an excellent process strategy, leading to a high level of rSpaA expression (about 25 % of total cellular protein content) in a short period of time.
本工作报道了来自猪丹毒丝菌 SpaA 蛋白的 42kDa 片段的克隆、表达和纯化,该片段是猪丹毒亚单位疫苗的主要候选抗原。还研究了使用自动诱导方案来提高重组大肠杆菌培养物中异源蛋白的表达。在不同诱导条件下评估了细胞生长模式和代谢产物的形成。His 标记的蛋白作为包含体过表达,并在变性条件下通过单一色谱步骤进行纯化。自动诱导条件被证明是一种极好的工艺策略,可在短时间内实现高水平的 rSpaA 表达(约占总细胞蛋白含量的 25%)。