Chemical complementation: A reaction-independent genetic assay for enzyme catalysis Academic Article uri icon

Overview

MeSH Major

  • Cephalosporinase
  • Enterobacter cloacae
  • Transcription, Genetic

abstract

  • A high-throughput assay for enzyme activity has been developed that is reaction independent. In this assay, a small-molecule yeast three-hybrid system is used to link enzyme catalysis to transcription of a reporter gene in vivo. Here we demonstrate the feasibility of this approach by using a well-studied enzyme-catalyzed reaction, cephalosporin hydrolysis by the Enterobacter cloacae P99 cephalosporinase (beta-lactam hydrolase, EC ). We show that the three-hybrid system can be used to read out cephalosporinase activity in vivo as a change in the level of transcription of a lacZ reporter gene and that the wild-type cephalosporinase can be isolated from a pool of inactive mutants by using a lacZ screen. The assay has been designed so that it can be applied to different chemical reactions without changing the components of the three-hybrid system. A reaction-independent high-throughput assay for protein function should be a powerful tool for protein engineering and enzymology, drug discovery, and proteomics.

publication date

  • December 24, 2002

Research

keywords

  • Academic Article

Identity

Language

  • eng

PubMed Central ID

  • PMC139179

Digital Object Identifier (DOI)

  • 10.1073/pnas.262420099

PubMed ID

  • 12482929

Additional Document Info

start page

  • 16537

end page

  • 42

volume

  • 99

number

  • 26