High-throughput screening for soluble recombinant expressed kinases in Escherichia coli and insect cells
High-throughput screening for soluble recombinant expressed kinases in Escherichia coli and insect cells
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DOI：
10.1016/j.pep.2004.03.003
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发表时间：
2004-07-01
影响因子：
1.6
通讯作者：
Kim, WM
中科院分区：
文献类型：
--
作者：
Chambers, SP;Austen, DA;Kim, WM
我们已经构建了一个双表达载体，用于在大肠杆菌和昆虫细胞中生产重组蛋白。在该载体中，杆状病毒多角体蛋白启动子位于噬菌体T7启动子和lac操纵子的上游。该载体被命名为pBEV，专门设计用于利用两种宿主都能提供的优势。该载体还有助于一站式克隆，从而简化了自动化表达过程，并开发了用于蛋白质表达的高通量方法。利用多系统载体pBEV，开发了一种高通量方法，在深孔块中表达并在微量滴定板中纯化，从而能够鉴定大肠杆菌和昆虫细胞中的表达和溶解度。在本研究中，我们利用pBEV成功地表达和纯化了多种人激酶。大肠杆菌和昆虫细胞。我们的研究结果验证了表达筛选作为一种策略，以快速筛选蛋白质，确定最佳的表达系统和生产条件。(C)2004年爱思唯尔公司All rights reserved.
We have constructed a dual expression vector for the production of recombinant proteins in both Escherichia coli and insect cells. In this vector, the baculoviral polyhedrin promoter was positioned upstream of the bacteriophage T7 promoter and the lac operator. This vector, designated pBEV, was specifically designed to exploit the advantages that both hosts would provide. This vector also facilitates one-stop cloning, thereby simplifying the expression process for automation, and the development of a high-throughput method for protein expression. Utilizing the multi-system vector pBEV, a high-throughput process was developed with expression in deep-well blocks and purification in micro-titer plates enabling the identification of expression and solubility in both E coli and insect cells. In this study, using pBEV, we have successfully expressed and purified multiple human kinases produced in E. coli and insect cells. Our results validate expression screening as a strategy to rapidly triage proteins identifying the optimum expression system and conditions for production. (C) 2004 Elsevier Inc. All rights reserved.