Traxlmayr Michael W, Faissner Maximilian, Stadlmayr Gerhard, Hasenhindl Christoph, Antes Bernhard, Rüker Florian, Obinger Christian
University of Natural Resources and Life Sciences, Vienna, Austria.
Biochim Biophys Acta. 2012 Apr;1824(4):542-9. doi: 10.1016/j.bbapap.2012.01.006. Epub 2012 Jan 20.
We have constructed IgG1-Fc scaffolds with increased thermal stability by directed evolution and yeast surface display. As a basis a new selection strategy that allowed the application of yeast surface display for screening of stabilizing mutations in proteins of already high intrinsic thermal stability and T(m)-values up to 85°C was developed. Besides library construction by error prone PCR, strong heat stress at 79°C for 10min and screening for well-folded proteins by FACS, sorting rounds had to include an efficient plasmid DNA isolation step for amplification and further transfection. We describe the successful application of this experimental setup for selection of 17 single, double and triple IgG1-Fc variants of increased thermal stability after four selection rounds. The recombinantly produced homodimeric proteins showed a wild-type-like elution profile in size exclusion chromatography as well as content of secondary structures. Moreover, the kinetics of binding of FcRn, CD16a and Protein A to the engineered Fc-molecules was very similar to the wild-type protein. These data clearly demonstrate the importance and efficacy of the presented strategy for selection of stabilizing mutations in proteins of high intrinsic stability within reasonable time.
我们通过定向进化和酵母表面展示构建了具有更高热稳定性的IgG1-Fc支架。在此基础上，开发了一种新的筛选策略，该策略允许应用酵母表面展示来筛选具有高固有热稳定性且T(m)值高达85°C的蛋白质中的稳定突变。除了通过易错PCR构建文库、在79°C下进行10分钟的强热应激以及通过荧光激活细胞分选术筛选折叠良好的蛋白质外，分选轮次还必须包括用于扩增和进一步转染的高效质粒DNA分离步骤。我们描述了该实验设置在四轮筛选后成功应用于选择17种具有更高热稳定性的单、双和三IgG1-Fc变体。重组产生的同二聚体蛋白在尺寸排阻色谱中显示出类似野生型的洗脱谱以及二级结构含量。此外，FcRn、CD16a和蛋白A与工程化Fc分子的结合动力学与野生型蛋白非常相似。这些数据清楚地证明了所提出的策略在合理时间内筛选高固有稳定性蛋白质中稳定突变的重要性和有效性。
