Faculty of Medicine and Dentistry, Membrane Protein Disease Research Group, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
Protein Sci. 2013 Apr;22(4):425-33. doi: 10.1002/pro.2223. Epub 2013 Feb 21.
The overexpression of milligram quantities of protein remains a key bottleneck in membrane protein structural biology. A challenge of particular difficulty has been the overproduction of eukaryotic membrane proteins. In order to cope with the frequently poor expression levels associated with these challenging proteins, it is often necessary to screen a large number of homologues to find a well expressing clone. To facilitate this process using the heterologous, eukaryotic expression host Pichia pastoris, we have developed a simple fluorescent induction plate-screening assay that allows for the rapid detection of well expressing clones of eukaryotic membrane proteins that have been fused to GFP. Using a eukaryotic membrane protein known to express well in P. pastoris (human aquaporin 4) and homologues of the ER associated membrane protein phosphatidylethanolamine N-methyltransferase (PEMT), we demonstrate that when a large number of clones are screened, a small number of highly expressing "jackpot" clones can be isolated. A jackpot PEMT clone resulted in 5 mg/L yield after purification. The method allows for the facile simultaneous screening of hundreds of clones providing an alternate to in-culture screening and will greatly accelerate the search for overexpressing eukaryotic membrane proteins.
大量表达蛋白质仍然是膜蛋白结构生物学的一个关键瓶颈。一个特别困难的挑战是真核膜蛋白的过度生产。为了应对这些具有挑战性的蛋白质通常与较差的表达水平相关的问题，通常需要筛选大量的同源物以找到表达良好的克隆。为了利用异源真核表达宿主毕赤酵母来促进这个过程，我们开发了一种简单的荧光诱导平板筛选检测法，该方法可以快速检测融合 GFP 的真核膜蛋白的表达良好的克隆。使用在毕赤酵母中表达良好的一种真核膜蛋白（人水通道蛋白 4）和内质网相关膜蛋白磷酸乙醇胺 N-甲基转移酶（PEMT）的同源物，我们证明了当筛选大量克隆时，可以分离出少数高表达的“ jackpot”克隆。一个 jackpot PEMT 克隆经纯化后产量达到 5mg/L。该方法可以轻松地同时筛选数百个克隆，为在培养物中筛选提供了替代方法，并将极大地加快寻找过表达的真核膜蛋白的速度。
