Affordable Access

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

Authors
Type
Published Article
Journal
Proceedings of the National Academy of Sciences of the United States of America
Publication Date
Volume
102
Issue
32
Pages
11224–11229
Identifiers
PMID: 16061816
Source
Medline

Abstract

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 10(6) 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.

There are no comments yet on this publication. Be the first to share your thoughts.

Statistics

Seen <100 times
0 Comments