Expression levels of transdominant peptides and proteins in <i>Saccharomyces cerevisiae</i>

Sandrock, Tanya; Poritz, Mark A.; Kim, Marianne; Feldhaus, Michael J.; Roth, Bruce L.; Caponigro, Giordano; Kamb, Alexander

doi:10.1002/yea.795

Cited by 7 publications

(3 citation statements)

References 16 publications

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“…However, a peptide selected in the context of the amino terminal portion of GFP functioned effectively when this GFP segment was removed and the peptide was grafted directly onto lexA. Due to differences in expression levels among C-terminal and internal GFP peptide constructs, we did not consider it useful to pursue more quantitative transactivation studies [22,27]. …”

Section: Discussionmentioning

confidence: 99%

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Section: Discussionmentioning

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“…They discovered peptides that inhibited cell growth and found that the phenotype could be suppressed by overexpression of ThyA protein. Peptide expression has also been used to knock out protein function and dissect signal transduction pathways in eukaryotic cells (5,7,9,13,17,18,41).…”

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

Benson¹,

Gottlin²,

Christensen³

et al. 2003

Antimicrob Agents Chemother

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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 growthinhibited 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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“…GFP has also been used as a scaffold to promote higher levels of expression of peptide aptamers [23][24][25][26]. GFP has also been used as a scaffold to promote higher levels of expression of peptide aptamers [23][24][25][26].…”

Section: Scaffold Proteins For the Presenta-tion Of Peptide Aptamersmentioning

confidence: 99%

Peptide Aptamer Libraries

Borghouts¹,

Künz²,

Groner³

2008

CCHTS

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Peptide aptamers are molecules that bind to protein targets and are able to interfere with their functions. In the past, important achievements have been made using such peptide aptamers in different approaches and for various purposes. Peptide aptamers are comprised of a variable peptide region of 8 to 20 amino acids in length, which is displayed by a scaffold protein. An overview of the numerous scaffold proteins that have been investigated for their suitability to present peptide aptamers will be given. To identify peptide aptamers efficiently and specifically binding to a predetermined target, two eukaryotic systems have been used in multiple studies: a modified version of the Gal4 yeast-two-hybrid system and the optimized LexA interaction trap system. The two yeast systems are compared and the design of high-complexity peptide aptamer libraries for these systems is described. Although the yeast-two-hybrid system is based on intracellular interactions mammalian screens, performed in cell culture experiments, are sometimes preferred or required. We will give an overview of the mammalian selection systems available, which are based on the expression of peptide aptamers in retroviral or lentiviral vectors. We will show that the isolation and use of peptide aptamers as inhibitors of individual signaling components represents a new challenge for drug development.

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Expression levels of transdominant peptides and proteins in Saccharomyces cerevisiae

Cited by 7 publications

References 16 publications

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

Peptide Aptamer Libraries

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