Vernet Erik, Kotzsch Alexander, Voldborg Bjørn, Sundström Michael
The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, 2200 Copenhagen N, Denmark.
Protein Expr Purif. 2011 May;77(1):104-11. doi: 10.1016/j.pep.2010.11.016. Epub 2010 Dec 2.
Soluble expression of proteins in a relevant form for functional and structural investigations still often remains a challenge. Although many biochemical factors are known to affect solubility, a thorough investigation of yield-limiting factors is normally not feasible in high-throughput efforts. Here we present a screening strategy for expression of biomedically relevant proteins in Escherichia coli using a panel of six different genetic variations. These include engineered strains for rare codon supplementation, increased disulfide bond formation in the cytoplasm and novel vectors for secretion to the periplasm or culture medium. Combining these variants with expression construct truncations design, we report on parallel cloning and expression of more than 300 constructs representing 24 selected proteins; including full-length variants of human growth factors, interleukins and growth factor binding proteins. This rapid screening approach appears highly suitable for high-throughput efforts targeting either large sets of proteins or more focused investigations regarding individual high-profile targets.
以适合功能和结构研究的相关形式实现蛋白质的可溶性表达仍然常常是一项挑战。尽管已知许多生化因素会影响溶解度，但在高通量研究中全面调查产量限制因素通常是不可行的。在此，我们展示了一种利用六种不同基因变异组合在大肠杆菌中表达生物医学相关蛋白质的筛选策略。这些变异包括用于补充稀有密码子的工程菌株、增强细胞质中二硫键形成的菌株以及用于分泌到周质或培养基中的新型载体。将这些变异与表达构建体截短设计相结合，我们报告了代表24种选定蛋白质的300多个构建体的平行克隆和表达；包括人类生长因子、白细胞介素和生长因子结合蛋白的全长变体。这种快速筛选方法似乎非常适合针对大量蛋白质的高通量研究或针对个别备受关注靶点的更有针对性的研究。
