Doyle Sharon A
Proteomics Group, DOE Joint Genome Institute, Walnut Creek, CA, USA.
Methods Mol Biol. 2005;310:115-21. doi: 10.1007/978-1-59259-948-6_8.
Protein expression and purification have traditionally been time-consuming, case-specific endeavors, and are considered to be the greatest bottlenecks in most proteomics pipelines. Escherichia coli (E. coli) is the most convenient and cost-effective host, although optimal conditions for the expression of different proteins vary widely. Proteins vary in their structural stability, solubility, and toxicity in this environment, resulting in differing rates of protein degradation, formation into insoluble inclusion bodies, and cell death, thus affecting the amount of soluble protein that can be obtained from E. coli grown in culture. To take full advantage of a variety of strategies developed to improve the expression of soluble protein in E. coli, an easy, rapid means to test many growth parameters is necessary. This chapter describes a dot-blot expression screen to test the effects of growth and induction parameters on the yield of soluble protein. The expression screen is used to detect hexahistidine-tagged proteins expressed in E. coli; however, it is adaptable for the detection of other affinity tags or fusion partners that have suitable antibodies available. In this example, induction time and temperature are tested; however, it can be used to test additional parameters, such as affinity tag type and placement, E. coli host type, and growth medium formulations.
传统上，蛋白质表达和纯化是耗时且针对具体情况的工作，被认为是大多数蛋白质组学流程中的最大瓶颈。大肠杆菌是最便捷且性价比高的宿主，尽管不同蛋白质表达的最佳条件差异很大。在这种环境中，蛋白质在结构稳定性、溶解性和毒性方面存在差异，导致蛋白质降解速率、形成不溶性包涵体以及细胞死亡的情况各不相同，从而影响从培养的大肠杆菌中可获得的可溶性蛋白质的量。为了充分利用为提高大肠杆菌中可溶性蛋白质表达而开发的各种策略，需要一种简单、快速的方法来测试多种生长参数。本章介绍一种斑点印迹表达筛选方法，以测试生长和诱导参数对可溶性蛋白质产量的影响。该表达筛选用于检测在大肠杆菌中表达的六聚组氨酸标签蛋白；然而，它适用于检测具有合适可用抗体的其他亲和标签或融合伙伴。在这个例子中，测试了诱导时间和温度；然而，它可用于测试其他参数，如亲和标签类型和位置、大肠杆菌宿主类型以及生长培养基配方。
