Quartley Erin, Alexandrov Andrei, Mikucki Maryann, Buckner Frederick S, Hol Wim G, DeTitta George T, Phizicky Eric M, Grayhack Elizabeth J
Center for Pediatric Biomedical Research, University of Rochester Medical School, Rochester, NY 14642, USA.
J Struct Funct Genomics. 2009 Sep;10(3):233-47. doi: 10.1007/s10969-009-9068-9. Epub 2009 Aug 22.
High level expression of many eukaryotic proteins for structural analysis is likely to require a eukaryotic host since many proteins are either insoluble or lack essential post-translational modifications when expressed in E. coli. The well-studied eukaryote Saccharomyces cerevisiae possesses several attributes of a good expression host: it is simple and inexpensive to culture, has proven genetic tractability, and has excellent recombinant DNA tools. We demonstrate here that this yeast exhibits three additional characteristics that are desirable in a eukaryotic expression host. First, expression in yeast significantly improves the solubility of proteins that are expressed but insoluble in E. coli. The expression and solubility of 83 Leishmania major ORFs were compared in S. cerevisiae and in E. coli, with the result that 42 of the 64 ORFs with good expression and poor solubility in E. coli are highly soluble in S. cerevisiae. Second, the yield and purity of heterologous proteins expressed in yeast is sufficient for structural analysis, as demonstrated with both small scale purifications of 21 highly expressed proteins and large scale purifications of 2 proteins, which yield highly homogeneous preparations. Third, protein expression can be improved by altering codon usage, based on the observation that a codon-optimized construct of one ORF yields three-fold more protein. Thus, these results provide direct verification that high level expression and purification of heterologous proteins in S. cerevisiae is feasible and likely to improve expression of proteins whose solubility in E. coli is poor.
由于许多蛋白质在大肠杆菌中表达时要么不溶，要么缺乏必要的翻译后修饰，因此对许多用于结构分析的真核蛋白质进行高水平表达可能需要真核宿主。经过充分研究的真核生物酿酒酵母具有作为良好表达宿主的几个特性：培养简单且成本低廉，已证明具有遗传易处理性，并且拥有出色的重组DNA工具。我们在此证明，这种酵母展现出了真核表达宿主所期望的另外三个特性。首先，在酵母中表达能显著提高那些在大肠杆菌中表达但不溶的蛋白质的溶解度。对83个利什曼原虫主要开放阅读框（ORF）在酿酒酵母和大肠杆菌中的表达及溶解度进行了比较，结果是在大肠杆菌中表达良好但溶解度差的64个ORF中有42个在酿酒酵母中高度可溶。其次，酵母中表达的异源蛋白质的产量和纯度足以用于结构分析，对21种高表达蛋白质的小规模纯化以及2种蛋白质的大规模纯化都证明了这一点，纯化后得到的是高度均一的制剂。第三，基于一个观察结果，即一个ORF的密码子优化构建体产生的蛋白质多三倍，通过改变密码子使用可以提高蛋白质表达。因此，这些结果直接证实了在酿酒酵母中高水平表达和纯化异源蛋白质是可行的，并且可能改善在大肠杆菌中溶解度差的蛋白质的表达。
