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Antimicrobial Agents and Chemotherapy, September 2003, p. 2875-2881, Vol. 47, No. 9
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.9.2875-2881.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Intracellular Expression of Peptide Fusions for Demonstration of Protein Essentiality in Bacteria

R. Edward Benson, Elizabeth B. Gottlin, Dale J. Christensen, and Paul T. Hamilton^*

Karo Bio USA, Inc., Durham, NC 27703

Received 16 December 2002/ Returned for modification 31 March 2003/ Accepted 6 June 2003

We describe a "protein knockout" technique that can be used to identify essential proteins in bacteria. This technique uses phage display to select peptides that bind specifically to purified target proteins. The peptides are expressed intracellularly and cause inhibition of growth when the protein is essential. In this study, peptides that each specifically bind to one of seven essential proteins were identified by phage display and then expressed as fusions to glutathione S-transferase in Escherichia coli. Expression of peptide fusions directed against E. coli DnaN, LpxA, RpoD, ProRS, SecA, GyrA, and Era each dramatically inhibited cell growth. Under the same conditions, a fusion with a randomized peptide sequence did not inhibit cell growth. In growth-inhibited cells, inhibition could be relieved by concurrent overexpression of the relevant target protein but not by coexpression of an irrelevant protein, indicating that growth inhibition was due to a specific interaction of the expressed peptide with its target. The protein knockout technique can be used to assess the essentiality of genes of unknown function emerging from the sequencing of microbial genomes. This technique can also be used to validate proteins as drug targets, and their corresponding peptides as screening tools, for discovery of new antimicrobial agents.

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* Corresponding author. Mailing address: Karo Bio USA, Inc., 4222 Emperor Blvd., Suite 560, Durham, NC 27703. Phone: (919) 474-8888 ext. 13. Fax: (919) 474-0103. E-mail: paul.hamilton{at}karobio.com.

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Antimicrobial Agents and Chemotherapy, September 2003, p. 2875-2881, Vol. 47, No. 9
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.9.2875-2881.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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