Automated expression and solubility screening of His-tagged proteins in 96-well format
Automated expression and solubility screening of His-tagged proteins in 96-well format
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DOI：
10.1016/j.ab.2005.07.039
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发表时间：
2005-11-01
影响因子：
2.9
通讯作者：
Bignon, C
中科院分区：
文献类型：
--
作者：
Vincentelli, R;Canaan, S;Bignon, C
结构基因组学对筛选重组蛋白表达和溶解度的灵敏、高通量方法的需求日益增长。最初，紧急解决方案是使用立即可用的技术，如手工裂解表达细胞，然后通过凝胶电泳分析蛋白质表达。然而，这些手工方法很快被证明不适合后基因组学的高通量需求，现在人们普遍认为，这个问题的长期解决方案将基于自动化，工业标准格式的实验，以及缩小样品和耗材。根据这一共识，我们建立了一种基于点印迹技术的全自动方法，并使用96孔格式消耗品，通过免疫检测评估大肠杆菌中表达的总和可溶性重组组氨酸（His）标记蛋白的数量。该方法从收获表达细胞开始，以每升培养物中重组蛋白的毫克数显示溶解度/表达结果结束，使用三色代码辅助分析。该程序一次可以自主处理160个独立的文化。(C) 2005爱思唯尔公司版权所有。
A growing need for sensitive and high-throughput methods for screening the expression and solubility of recombinant proteins exists in structural genomics. Originally, the emergency solution was to use immediately available techniques such as manual lysis of expression cells followed by analysis of protein expression by gel electrophoresis. However, these handmade methods quickly proved to be unfit for the high-throughput demand of postgenomics, and it is now generally accepted that the long-term solution to this problem will be based on automation, on industrial standard-formatted experiments, and on downsizing samples and consumables. In agreement with this consensus, we have set up a fully automated method based on a dot-blot technology and using 96-well format consumables for assessing by immunodetection the amount of total and soluble recombinant histidine (His)-tagged proteins expressed in Escherichia coli. The method starts with the harvest of expression cells and ends with the display of solubility/expression results in milligrams of recombinant protein per liter of culture using a three-color code to assist analysis. The program autonomously processes 160 independent cultures at a time. (C) 2005 Elsevier Inc. All rights reserved.