Chemical Complementation: a Reaction-independent Genetic Assay for Enzyme Catalysis

Overview

Journal Proc Natl Acad Sci U S A

Specialty Science

Date 2002 Dec 17

PMID 12482929

Citations 24

Authors

Kathleen Baker

Colleen Bleczinski

Hening Lin

Gilda Salazar-Jimenez

Debleena Sengupta

Sonja Krane

Virginia W Cornish

Affiliations

Soon will be listed here.

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.

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