Deepak Selvam , Anish D'silva , Arun Panchapakesan , Yuvrajsinh Gohil , Jayendra Singh , Luke Elizabeth Hanna , Udaykumar Ranga
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引用次数: 0
摘要
在异源宿主中高效表达功能蛋白质已成为现代生物技术和生物医学研究的关键重点。为此,人们正在探索大肠杆菌的多种替代品来表达重组蛋白。L. lactis 是一种革兰氏阳性生物,可避免蛋白质纯化过程中的内毒素去除步骤。我们在此报告在乳球菌系统中优化 HIV-1 Tat(一种众所周知的难表达蛋白质)的表达。我们在两种不同的乳球菌菌株中评估了五种不同的启动子,并考察了 pH 值、葡萄糖和诱导时间对 Tat 产量和纯度的影响。最后,重组 Tat 在 HLM-1 报告细胞中具有转激活 HIV-1 启动子的功能。我们的工作为今后在乳酸乳球菌中表达毒性蛋白的工作提供了一个支架。
Efficient expression of functional proteins in heterologous hosts has become the pivotal focus of modern biotechnology and biomedical research. To this end, multiple alternatives to E. coli are being explored for recombinant protein expression. L. lactis, being a gram-positive organism, circumvents the need for an endotoxin removal step during protein purification. We report here the optimisation of the expression of HIV-1 Tat, a notoriously difficult protein, in Lactococcus lactis system. We evaluated five different promoters in two different Lactococcus lactis strains and examined the effect of pH, glucose, and induction time on the yield and purity of Tat. Finally, the recombinant Tat was functionally competent in transactivating the HIV-1 promoter in HLM-1 reporter cells. Our work provides a scaffold for future work on the expression of toxic proteins in Lactococcus lactis.
期刊介绍:
Protein Expression and Purification is an international journal providing a forum for the dissemination of new information on protein expression, extraction, purification, characterization, and/or applications using conventional biochemical and/or modern molecular biological approaches and methods, which are of broad interest to the field. The journal does not typically publish repetitive examples of protein expression and purification involving standard, well-established, methods. However, exceptions might include studies on important and/or difficult to express and/or purify proteins and/or studies that include extensive protein characterization, which provide new, previously unpublished information.