Directed evolution of protein switches and their application to the creation of ligand-binding proteins

被引:164
作者
Guntas, G [1 ]
Mansell, TJ [1 ]
Kim, JR [1 ]
Ostermeier, M [1 ]
机构
[1] Johns Hopkins Univ, Dept Chem & Biomol Engn, Baltimore, MD 21218 USA
关键词
allostery; beta-lactamase; maltose binding protein;
D O I
10.1073/pnas.0502673102
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
We describe an iterative approach for creating protein switches involving the in vitro recombination of two nonhomologous genes. We demonstrate this approach by recombining the genes coding for TEM1 beta-lactamase (BLA) and the Escherichia coli maltose binding protein (MBP) to create a family of MBP-BLA hybrids in which maltose is a positive or negative effector of beta-lactam hydrolysis. Some of these MBP-BLA switches were effectively "on-off" in nature, with maltose altering catalytic activity by as much as 600-fold. The ability of these switches to confer an effector-dependent growth/no growth phenotype to E. coli cells was exploited to rapidly identify, from a library of 4 x 106 variants, MBP-BLA switch variants that respond to sucrose as the effector. The transplantation of these mutations into wild-type MBP converted MBP into a "sucrose-binding protein," illustrating the switches potential as a tool to rapidly identify ligand-binding proteins.
引用
收藏
页码:11224 / 11229
页数:6
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