Green fluorescent protein-based expression screening of membrane proteins in Escherichia coli.
Green fluorescent protein-based expression screening of membrane proteins in Escherichia coli.
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DOI：
10.3791/52357
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发表时间：
2015-01-06
期刊：
影响因子：
--
通讯作者：
Owens RJ
中科院分区：
文献类型：
--
作者：
Bird LE;Rada H;Verma A;Gasper R;Birch J;Jennions M;Lӧwe J;Moraes I;Owens RJ
用于结构和功能研究的重组膜蛋白的生产在技术上仍然具有挑战性，这是由于低水平的表达和许多膜蛋白一旦溶解在洗涤剂中固有的不稳定性。描述了一种方案，其结合了膜蛋白作为GFP融合物的连接独立克隆与通过GFP荧光检测的大肠杆菌中的表达。这使得能够构建和表达筛选多个膜蛋白/变体，以鉴定适合于进一步投入时间和精力的候选物。GFP报告基因通过在SDS聚丙烯酰胺凝胶电泳（SDS-PAGE）后观察GFP荧光用于表达的初步筛选。选择既显示高表达水平又具有最小降解的膜蛋白用于二次筛选，所述最小降解由不存在游离GFP指示。将这些构建体按比例缩放，并制备总膜级分并溶解在四种不同的洗涤剂中。在超离心以除去去污剂不溶性物质后，通过荧光检测尺寸排阻色谱法（FSEC）分析裂解物。通过GFP荧光监测尺寸排阻分布提供了关于膜蛋白在不同洗涤剂中的单分散性和完整性的信息。蛋白质：具有对称峰的洗涤剂组合，几乎没有或没有游离GFP和最小聚集，是随后纯化的候选物。利用上述方法，在E.分析了47种不同细菌的SED（形状、延伸、分裂和孢子形成）蛋白在大肠杆菌中的表达。这些蛋白质通常具有十个跨膜结构域，并且对于细胞分裂是必需的。结果表明，在E.在表达水平和GFP融合蛋白的完整性方面，大肠杆菌是高度可变的。该实验确定了一个子集进行进一步研究。
The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.
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影响因子：
5.7
作者：
Sonoda, Yo;Newstead, Simon;Hu, Nien-Jen;Alguel, Yilmaz;Nji, Emmanuel;Beis, Konstantinos;Yashiro, Shoko;Lee, Chiara;Leung, James;Cameron, Alexander D.;Byrne, Bernadette;Iwata, So;Drew, David
通讯作者：
Drew, David
影响因子：
14.8
作者：
Drew, David;Newstead, Simon;Iwata, So
通讯作者：
Iwata, So
影响因子：
8
作者：
Wallin, E;von Heijne, G
通讯作者：
von Heijne, G
影响因子：
5.6
作者：
Schlegel, Susan;Lofblom, John;de Gier, Jan-Willem
通讯作者：
de Gier, Jan-Willem
DOI：
10.1016/j.bbagen.2013.02.001
发表时间：
2013-06-01
影响因子：
3
作者：
Low, Christian;Moberg, Per;Nordlund, Par
通讯作者：
Nordlund, Par