Ho Franz Y, Poolman Bert
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute & Zernike Institute for Advanced Materials, University of Groningen, Groningen, The Netherlands.
Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute & Zernike Institute for Advanced Materials, University of Groningen, Groningen, The Netherlands.
Methods Enzymol. 2015;556:3-21. doi: 10.1016/bs.mie.2015.01.003. Epub 2015 Mar 20.
A major bottleneck in the characterization of membrane proteins is low yield of functional protein in recombinant expression. Microorganisms are widely used for recombinant protein production, because of ease of cultivation and high protein yield. However, the target proteins do not always obtain their native conformation and may end up in a nonfunctional state, in insoluble aggregates. For screening of functional protein, it is thus important to readily discriminate aggregated, mistargeted protein from globally well-folded, membrane-inserted protein. We developed a robust strategy for expression screening of functional proteins in bacteria, which is based on directed evolution. In this strategy, the C-terminus of the target membrane protein is tagged with two additional protein domains in tandem. The first one is green fluorescent protein (GFP), which functions as a reporter of the global folding state of the fusion protein. The other one is the erythromycin resistance protein (23S ribosomal RNA adenine N-6 methyltransferase, ErmC), which confers a means to select for enhanced expression. By gradually increasing the antibiotic concentration in the medium, we force the cells to evolve in a way that allows more functional target-GFP-ErmC to be expressed. The acquired genomic mutations can be generic or membrane protein specific. This strategy is readily adopted for the expression of any protein and ultimately yields a wealth of genomic data that may provide insight into the factors that limit the production of given classes or types of proteins.
膜蛋白表征的一个主要瓶颈是重组表达中功能蛋白的产量低。由于易于培养和高蛋白产量，微生物被广泛用于重组蛋白生产。然而，目标蛋白并不总是能获得其天然构象，最终可能处于非功能状态，形成不溶性聚集体。因此，对于功能性蛋白的筛选，重要的是要能够轻松地区分聚集的、靶向错误的蛋白与整体折叠良好、插入膜中的蛋白。我们开发了一种基于定向进化的强大策略，用于在细菌中筛选功能性蛋白的表达。在该策略中，目标膜蛋白的C末端串联标记有两个额外的蛋白结构域。第一个是绿色荧光蛋白（GFP），它作为融合蛋白整体折叠状态的报告分子。另一个是红霉素抗性蛋白（23S核糖体RNA腺嘌呤N-6甲基转移酶，ErmC），它提供了一种选择增强表达的方法。通过逐渐增加培养基中的抗生素浓度，我们迫使细胞以一种允许表达更多功能性目标-GFP-ErmC的方式进化。获得的基因组突变可以是通用的，也可以是膜蛋白特异性的。该策略很容易应用于任何蛋白的表达，并最终产生大量的基因组数据，这些数据可能有助于深入了解限制特定类别或类型蛋白生产的因素。
