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A high-throughput microtiter plate-based screening method for the detection of full-length recombinant proteins

机译：基于高通量微量滴定板的筛选方法，用于检测全长重组蛋白

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The Gram-negative bacterium Escherichia coli is an important host for the (heterologous) production of recombinant proteins. The development and optimization of a protocol to overproduce a desired protein in E. coli is often tedious. A novel high-throughput screening method based on the Luminex(R) xMAP(TM) bead technology was developed allowing a rapid evaluation of a certain expression strategy. A variant of green fluorescent protein (GFPuv) from Aequorea victoria was used as a reporter to establish the methodology. The N-terminus and the C-terminus of GFPuv were engineered to contain a HiS(6)- and an HA-tag (YPYDVPDYA), respectively. The double-tagged protein was loaded onto Luminex-microspheres via its His(6)-tag, the presence of the HA-tag was verified using an anti-HA antibody. High-throughput detection of full-length proteins (containing both tags) on the beads was performed using an automated Luminex 100IS analyzer. The results were compared to results obtained by classical Western blot analysis. Comparison of the two methods revealed that the Luminex-based method is faster and more economical in detecting full-length (intact) soluble recombinant protein, allowing one to routinely screen a high number of parameters in gene expression experiments. As proof of concept, different protocols to overproduce double-tagged model eucaryotic proteins (human protein S6 kinase 1 and human tankyrase) in E. coli were monitored using the new approach. Relevant parameters for optimizing gene expression of the corresponding genes were rapidly identified using the novel high-throughput method. (C) 2008 Elsevier Inc. All rights reserved.
机译：革兰氏阴性细菌大肠杆菌是重组蛋白（异源）生产的重要宿主。在大肠杆菌中过量生产所需蛋白质的方案的开发和优化通常是乏味的。开发了一种基于Luminex（R）xMAP（TM）珠技术的新型高通量筛选方法，可以快速评估某种表达策略。来自维多利亚水母（Aequorea victoria）的绿色荧光蛋白（GFPuv）的变体用作报告基因，建立了方法。 GFPuv的N末端和C末端经过工程改造，分别包含一个HiS（6）-和一个HA标签（YPYDVPDYA）。双标签的蛋白质通过其His（6）-标签加载到Luminex微球上，使用抗HA抗体验证了HA-标签的存在。使用自动化Luminex 100IS分析仪对珠子上的全长蛋白质（包含两个标签）进行高通量检测。将结果与通过经典蛋白质印迹分析获得的结果进行比较。两种方法的比较表明，基于Luminex的方法在检测全长（完整）可溶性重组蛋白时更快捷，更经济，从而使人们可以在基因表达实验中常规筛选大量参数。作为概念的证明，使用新方法监测了在大肠杆菌中过量产生双标签模型真核蛋白（人蛋白S6激酶1和人tankyrase）的不同方案。使用新颖的高通量方法可以快速确定优化相应基因的基因表达的相关参数。 （C）2008 Elsevier Inc.保留所有权利。

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