Jasniewski Jordane, Cailliez-Grimal Catherine, Gelhaye Eric, Revol-Junelles Anne-Marie
Laboratoire de Science et Génie Alimentaires (LSGA), Nancy-Université, 2 avenue de la Forêt de Haye, B.P. 172 F-54505 Vandoeuvre-lès-Nancy, France.
J Microbiol Methods. 2008 Apr;73(1):41-8. doi: 10.1016/j.mimet.2008.01.008. Epub 2008 Feb 6.
An optimization of the production and purification processes of carnobacteriocins Cbn BM1 and Cbn B2 from Carnobacterium maltaromaticum CP5, by heterologous expression in Escherichia coli is described. The genes encoding mature bacteriocin were cloned into an E. coli expression system and expressed as a fusion protein with a thermostable thioredoxin. Recombinant E. coli were cultivated following a fed-batch fermentation process with pH, temperature and oxygenation regulation. The overexpression of the fusion proteins was improved by replacing IPTG by lactose. The fusion proteins were purified by thermal coagulation followed by affinity chromatography. The thioredoxin fusion protein was removed by using CNBr instead of enterokinase and the carnobacteriocins were recovered by reverse-phase chromatography. These optimizations led us to produce up to 320 mg of pure protein per liter of culture, which is four to ten fold higher than what is described for other heterologous expression systems.
本文描述了通过在大肠杆菌中进行异源表达，对来自麦芽香鱼酒球菌CP5的肉杆菌素Cbn BM1和Cbn B2的生产和纯化工艺进行优化的过程。将编码成熟细菌素的基因克隆到大肠杆菌表达系统中，并作为与热稳定硫氧还蛋白的融合蛋白进行表达。重组大肠杆菌采用补料分批发酵工艺进行培养，并对pH、温度和氧合进行调节。用乳糖替代IPTG提高了融合蛋白的过表达。融合蛋白先通过热凝固然后通过亲和层析进行纯化。使用溴化氰而非肠激酶去除硫氧还蛋白融合蛋白，并通过反相层析回收肉杆菌素。这些优化使我们每升培养物能够生产高达320毫克纯蛋白，比其他异源表达系统的产量高四至十倍。
