CIS display: In vitro selection of peptides from libraries of protein-DNA complexes.
We describe the development of an in vitro library selection system (CIS display) that exploits the ability of a DNA replication initiator protein (RepA) to bind exclusively to the template DNA from which it has been expressed, a property called cis-activity. A diverse peptide library is created by ligation of DNA fragments of random sequence to a DNA fragment that encodes RepA. After in vitro transcription and translation, a pool of protein-DNA complexes is formed where each protein is stably associated with the DNA that encodes it. These complexes are amenable to the affinity selection of ligands to targets of interest. Here we show that RepA is a highly faithful cis-acting DNA-binding protein and demonstrate that libraries encoding >10(12) random 18-mer peptides can be constructed and used to isolate peptides that bind specifically to disparate targets. The use of DNA to encode the displayed peptides offers advantages over in vitro peptide display systems that use mRNA.
Odegrip
Richard
R
Isogenica Ltd., Babraham Hall, Babraham CB2 4AT, United Kingdom.
Coomber
David
D
Eldridge
Bill
B
Hederer
Rosemarie
R
Kuhlman
Philip A
PA
Ullman
Christopher
C
FitzGerald
Kevin
K
McGregor
Duncan
D
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004265:DNA Helicases; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D011506:Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015534:Trans-Activators
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D019151:Peptide Library; D010455:Peptides; D011506:Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; C045264:replication initiator protein; D004247:DNA; D004265:DNA Helicases