Directed evolution of stabilized IgG1-Fc scaffolds by application of strong heat shock to libraries displayed on yeast

详细信息	查看全文 | 推荐本文 |

• 作者：Michael W. Traxlmayr^a ; ^b ; Maximilian Faissner^a ; ^b ; Gerhard Stadlmayr^a ; ^b ; Christoph Hasenhindl^a ; ^b ; Bernhard Antes^d ; Florian Rü ; ker^a ; ^c ; Christian Obinger^a ; ^b ; ^{christian.obinger@boku.ac.at}
• 关键词：IgG1 ; immunoglobulin G class 1 ; Fcab ; antigen binding Fc protein ; Fc-wt ; recombinant wild-type Fc protein ; scTCR ; single-chain T-cell receptor ; MHC ; major histocompatibility complex ; CDC ; complement dependent cytotoxicity ; ADCC ; antibody dependent cell-me
• 刊名：Biochimica et Biophysica Acta - Proteins and Proteomics
• 出版年：2012
• 期刊代码：161_15709639
• 类别：bio
• 出版时间：April, 2012
• 卷：1824
• 期：4
• 页码：542-549
• 文件大小：1404 K

摘要

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 掳C was developed. Besides library construction by error prone PCR, strong heat stress at 79 掳C for 10 min 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.