Engineering High Affinity Protein–Protein Interactions Using a High-Throughput Microcapillary Array Platform
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- 作者：Sungwon LimBob Chen ; Mihalis S. Kariolis ; Ivan K. Dimov ; Thomas M. Baer ; Jennifer R. Cochran
- 刊名：ACS Chemical Biology
- 出版年：2017
- 出版时间：February 17, 2017
- 年：2017
- 卷：12
- 期：2
- 页码：336-341
- 全文大小：361K
- ISSN：1554-8937
文摘
Affinity maturation of protein–protein interactions requires iterative rounds of protein library generation and high-throughput screening to identify variants that bind with increased affinity to a target of interest. We recently developed a multipurpose protein engineering platform, termed μSCALE (Microcapillary Single Cell Analysis and Laser Extraction). This technology enables high-throughput screening of libraries of millions of cell-expressing protein variants based on their binding properties or functional activity. Here, we demonstrate the first use of the μSCALE platform for affinity maturation of a protein–protein binding interaction. In this proof-of-concept study, we engineered an extracellular domain of the Axl receptor tyrosine kinase to bind tighter to its ligand Gas6. Within 2 weeks, two iterative rounds of library generation and screening resulted in engineered Axl variants with a 50-fold decrease in kinetic dissociation rate, highlighting the use of μSCALE as a new tool for directed evolution.