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Fluorescent probe for high-throughput screening of membrane protein expression

机译:高通量筛选膜蛋白表达的荧光探针

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摘要

Screening of protein variants requires specific detection methods to assay protein levels and stability in crude mixtures. Many strategies apply fluorescence-detection size-exclusion chromatography (FSEC) using green fluorescent protein (GFP) fusion proteins to qualitatively monitor expression, stability, and monodispersity. However, GFP fusion proteins have several important disadvantages; including false-positives, protein aggregation after proteolytic removal of GFP, and reductions in protein yields without the GFP fusion. Here we describe a FSEC screening strategy based on a fluorescent multivalent NTA probe that interacts with polyhistidine-tags on target proteins. This method overcomes the limitations of GFP fusion proteins, and can be used to rank protein production based on qualitative and quantitative parameters. Domain boundaries of the human G-protein coupled adenosine A2a receptor were readily identified from crude detergent-extracts of a library of construct variants transiently produced in suspension-adapted HEK293-6E cells. Well expressing clones of MraY, an important bacterial infection target, could be identified from a library of 24 orthologs. This probe provides a highly sensitive tool to detect target proteins to expression levels down to 0.02 mg/L in crude lysate, and requires minimal amounts of cell culture.
机译:蛋白质变体的筛选需要特定的检测方法来测定粗混合物中的蛋白质水平和稳定性。许多策略都应用了使用绿色荧光蛋白(GFP)融合蛋白的荧光检测尺寸排阻色谱(FSEC),以定性地监测表达,稳定性和单分散性。然而,GFP融合蛋白具有几个重要的缺点。包括假阳性,蛋白水解去除GFP后的蛋白质聚集,以及没有GFP融合的蛋白质产量降低。在这里,我们描述了一种基于荧光多价NTA探针的FSEC筛选策略,该探针与目标蛋白上的多组氨酸标签相互作用。该方法克服了GFP融合蛋白的局限性,可用于基于定性和定量参数对蛋白质生产进行排名。从与悬浮液相适应的HEK293-6E细胞中瞬时产生的构建体变体文库的粗洗涤剂提取物中,很容易鉴定出人G蛋白偶联的腺苷A2a受体的结构域边界。可以从24个直系同源物的文库中鉴定出表达良好的重要细菌感染靶标MraY的克隆。该探针提供了一种高度灵敏的工具,可检测目标蛋白在粗裂解物中的表达水平低至0.02 mg / L,并且所需的细胞培养量最少。

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