Chemical complementation: A reaction-independent genetic assay for enzyme catalysis
- Kathleen Baker*,†,
- Colleen Bleczinski†,‡,
- Hening Lin‡,
- Gilda Salazar-Jimenez‡,
- Debleena Sengupta‡,
- Sonja Krane‡, and
- Virginia W. Cornish‡,§
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Edited by Charles R. Cantor, Sequenom, Inc., San Diego, CA, and approved October 23, 2002 (received for review July 15, 2002)
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 (β-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.
Footnotes
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↵ † K.B. and C.B. contributed equally to this work.
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↵ § To whom correspondence should be addressed at: Department of Chemistry, Columbia University, Havemeyer Hall, MC 3111, 3000 Broadway, New York, NY 10027. E-mail: vc114{at}columbia.edu.
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This paper was submitted directly (Track II) to the PNAS office.
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See commentary on page 16513.
- Abbreviations:
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Mtx, methotrexate
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Dex, dexamethasone
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DHFR, dihydrofolate reductase
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GR, glucocorticoid receptor
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X-Gal, 5-bromo-4-chloro-3-indolyl β-d-galactoside
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ONPG, o-nitrophenyl β-d-galactopyranoside
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ACLE, 7-amino-3-chlormethyl-3-cephem-4-carboxylic acid p-methoxybenzyl ester
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- Copyright © 2002, The National Academy of Sciences
