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

Gurkan Guntas, Thomas J. Mansell, Jin Kim, Marc Ostermeier

Research output: Contribution to journalArticle

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 β-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 β-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 × 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.

Original languageEnglish (US)
Pages (from-to)11224-11229
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume102
Issue number32
DOIs
StatePublished - Aug 9 2005

Fingerprint

Maltose-Binding Proteins
Carrier Proteins
Ligands
Proteins
Maltose
Sucrose
Lactams
Escherichia coli Proteins
Growth
Genetic Recombination
Genes
Hydrolysis
Transplantation
Escherichia coli
Phenotype
Mutation

Keywords

  • β-lactamase
  • Allostery
  • Maltose binding protein

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Directed evolution of protein switches and their application to the creation of ligand-binding proteins. / Guntas, Gurkan; Mansell, Thomas J.; Kim, Jin; Ostermeier, Marc.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 102, No. 32, 09.08.2005, p. 11224-11229.

Research output: Contribution to journalArticle

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