Geertsma Eric R, Poolman Bert
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, Netherlands Proteomics Centre, University of Groningen, The Netherlands.
Methods Mol Biol. 2010;601:17-38. doi: 10.1007/978-1-60761-344-2_2.
As the equivalent to gatekeepers of the cell, membrane transport proteins perform a variety of critical functions. Progress on the functional and structural characterization of membrane proteins is slowed due to problems associated with their (heterologous) overexpression. Often, overexpression fails or leads to aggregated material from which the production of functionally refolded protein is challenging. It is still difficult to predict whether a given membrane protein can be overproduced in a functional competent state. As a result, the most straightforward strategy to set up an overexpression system is to screen a multitude of conditions, including the comparison of homologues, type and location of (affinity) tags, and distinct expression hosts. Here, we detail methodology to rapidly establish and optimize (membrane) protein expression in Escherichia coli and Lactococcus lactis.
作为细胞的“看门人”，膜转运蛋白执行着多种关键功能。由于与膜蛋白（异源）过表达相关的问题，其功能和结构表征的进展较为缓慢。通常，过表达会失败，或者导致产生聚集物，从中生产功能重折叠的蛋白质具有挑战性。仍然难以预测给定的膜蛋白是否能够在功能上胜任的状态下过量生产。因此，建立过表达系统最直接的策略是筛选多种条件，包括同源物的比较、（亲和）标签的类型和位置以及不同的表达宿主。在这里，我们详细介绍了在大肠杆菌和乳酸乳球菌中快速建立和优化（膜）蛋白表达的方法。
