Intracellular expression of Peptide fusions for demonstration of protein essentiality in bacteria.
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.
Benson
R Edward
RE
Karo Bio USA, Inc, Durham, NC 27703, USA.
Gottlin
Elizabeth B
EB
Christensen
Dale J
DJ
Hamilton
Paul T
PT
D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology*; D015153:Blotting, Western; D003470:Culture Media; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism; D004278:DNA, Superhelical / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism; D005822:Genetic Vectors; D019151:Peptide Library; D010455:Peptides / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000235:genetics
D001426:Bacterial Proteins; D003470:Culture Media; D004269:DNA, Bacterial; D004278:DNA, Superhelical; D019151:Peptide Library; D010455:Peptides