CIS display: In vitro selection of peptides from libraries of protein–DNA complexes

Abstract

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.

Beyond the Natural Proteome

“…Adapted from Ashraf, M., Frigotto, L., Smith, M.E., Patel, S., Hughes, M.D., Poole, A.J., et al (2013) Adapted from Odegrip et al, 2004. Double-standed DNA is generated consisting of (in 5'  3' order) a promoter, the saturated library fused in-frame to repA, the CIS element and the ori sequence.…”

“…The resulting complex is screened via biopanning. Adapted from Odegrip, R., Coomber, D., Eldridge, B., Hederer, R., Kuhlman, P.A., Ullman, C. et al (2004) …”

“…Though useful for many applications, phage display is limited by transformation efficiency and cannot therefore be used to screen libraries of greater than 10 9 to 10 10 components (Odegrip, Coomber, Eldridge, Hederer, Kuhlman & Ullman et al, 2004).…”

“…Ultimately, through its cis activity, RepA protein provides the crucial physical linkage between genotype (DNA) and phenotype (protein; Figure 5). After each round of biopanning, DNA from the eluted complex is either amplified by PCR ready for the next round of biopanning, or sequenced to identify the interacting library component (Odegrip et al, 2004;Mathonet, Ioannou, Betley & Ullman, 2011).…”

“…CIS display has been used in antibody research (Odegrip et al, 2004), peptide maturation, ligand discovery including therapeutic peptides (Mathonet et al, 2011), and engineering of small WW scaffolds (small β-sheet motifs; Patel, Mathonet, Jaulent & Ullman, 2013). CIS display technology has also been used to identify 12-mer peptides resistant to thrombin, chymotrypsin and plasma proteases (Eldridge, Cooley, Odegrip, McGregor, FitzGerald & Ullman, 2009).…”