Department of Biomedical Sciences and Engineering, University of Nova Gorica (UNG), Glavni Trg 9 - SI-5261, Vipava, Slovenia.
Microb Cell Fact. 2013 Nov 1;12:101. doi: 10.1186/1475-2859-12-101.
The development of complementary technologies enabled the successful production of recombinant polypeptides in bacteria and opened to biology researchers new avenues as obtaining suitable amounts of proteins necessary for their experimental work became easy, fast, and inexpensive. Nevertheless, the recombinant approach remained somehow unpredictable, since many constructs resisted to apparent production or accumulated as aggregates. Several factors and physical/chemical conditions that could improve the accumulation of native-like protein were identified. At the same time, it was acknowledged that the outcome of most of them was erratic and that almost any protein required its own specific optimized set of conditions to achieve its correct folding. The attempt to understand the critical points specific for recombinant protein production missed the goal of setting universally useful protocols, but contributed to the increase of the rate of success by proposing always new empiric combinations.Nevertheless, the results published in the recent literature allow for a better comprehension of some key mechanisms controlling protein production in E. coli and could enable the elaboration of rational methodologies for improving the quantitative and qualitative features of the produced polypeptides. This result will be achieved when the identification of the limiting step that impairs the accomplishment of the native folding for any single construct will become straightforward. This minireview will discuss how factors such as the expression rate, the folding machinery, and the secretion efficiency may impact the final protein yields.
互补技术的发展使得重组多肽在细菌中的成功生产成为可能，为生物学研究人员开辟了新的途径，因为获得实验所需的大量合适蛋白质变得容易、快速且廉价。然而，重组方法仍然存在一定的不可预测性，因为许多构建体难以进行明显的生产或聚集形成聚集体。已经确定了几种可以提高类似天然蛋白质积累的因素和物理/化学条件。同时，人们也认识到，它们中的大多数的结果都是不稳定的，几乎任何蛋白质都需要自己特定的优化条件组合才能实现正确折叠。试图理解重组蛋白生产的具体关键点的尝试未能达到制定普遍有用方案的目标，但通过提出新的经验组合，有助于提高成功率。然而，最近文献中的研究结果使人们能够更好地理解控制大肠杆菌中蛋白质生产的一些关键机制，并能够为改善产生的多肽的定量和定性特征制定合理的方法学。当确定任何单个构建体中阻碍天然折叠完成的限制步骤变得直接时，就会实现这一结果。这篇综述将讨论表达率、折叠机制和分泌效率等因素如何影响最终的蛋白质产量。
