Korf Ulrike, Kohl Thorsten, van der Zandt Hans, Zahn Regina, Schleeger Simone, Ueberle Barbara, Wandschneider Silke, Bechtel Stephanie, Schnölzer Martina, Ottleben Holger, Wiemann Stefan, Poustka Annemarie
Molecular Genome Analysis, DKFZ, Heidelberg, Germany.
Proteomics. 2005 Sep;5(14):3571-80. doi: 10.1002/pmic.200401195.
Access to pure and soluble recombinant proteins is essential for numerous applications in proteome research, such as the production of antibodies, structural characterization of proteins, and protein microarrays. Through the German cDNA Consortium we have access to more than 1500 ORFs encoding uncharacterized proteins. Preparing a large number of recombinant proteins calls for the careful refinement and re-evaluation of protein purification tools. The expression and purification strategy should result in mg quantities of protein that can be employed in microarray-based assays. In addition, the experimental set-up should be robust enough to allow both automated protein expression screening and the production of the proteins on a mg scale. These requirements are best fulfilled by a bacterial expression system such as Escherichia coli. To develop an efficient expression strategy, 75 different ORFs were transferred into suitable expression vectors using the Gateway cloning system. Four different fusion tags (E. coli transcription-termination anti-termination factor (NusA), hexahistidine tag (6xHis), maltose binding protein (MBP) and GST) were analyzed for their effect on yield of induced fusion protein and its solubility, as determined at two different induction temperatures. Affinity-purified fusion proteins were confirmed by MALDI-TOF MS.
获得纯的和可溶的重组蛋白对于蛋白质组研究中的众多应用至关重要，例如抗体的生产、蛋白质的结构表征以及蛋白质微阵列。通过德国cDNA联盟，我们能够获取1500多个编码未鉴定蛋白质的开放阅读框（ORF）。制备大量重组蛋白需要对蛋白质纯化工具进行仔细的优化和重新评估。表达和纯化策略应能产生毫克级的蛋白质，可用于基于微阵列的分析。此外，实验设置应足够稳健，以允许自动蛋白质表达筛选和毫克规模的蛋白质生产。这些要求最好由细菌表达系统如大肠杆菌来满足。为了开发一种高效的表达策略，使用Gateway克隆系统将75个不同的开放阅读框转移到合适的表达载体中。分析了四种不同的融合标签（大肠杆菌转录终止抗终止因子（NusA）、六聚组氨酸标签（6xHis）、麦芽糖结合蛋白（MBP）和谷胱甘肽S-转移酶（GST））对诱导融合蛋白产量及其溶解度的影响，这是在两个不同的诱导温度下测定的。通过基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）对亲和纯化的融合蛋白进行了确认。
