Protocol abstract
Nature Protocols 2, - 1126 - 1133 (2007)
Published online: 3 May 2007 | doi:10.1038/nprot.2007.152
Subject Categories: Isolation, purification and separation | Genetic modification | Nucleic acid based molecular biology | Biochemistry and protein analysis
Split-intein mediated circular ligation used in the synthesis of cyclic peptide libraries in E. coli
Ali Tavassoli1 & Stephen J Benkovic2
Abstract
Recent advances in chemical biology and the advantages presented by in vivo screening have highlighted the need for a robust and flexible biologically synthesized small-molecule library. Herein we describe a method for the biosynthesis of cyclic peptide libraries of up to 108 members in Escherichia coli using split-intein circular ligation of peptides and proteins (SICLOPPS). The method utilizes split-intein chemistry to cyclize randomized peptide sequences. The cyclic peptide library can potentially be of any size and the peptide itself may contain unlimited random residues. However, the library size is limited by the transformation efficiency of E. coli and random residues are generally limited to five, but additional amino acids can be used in the cyclic peptide backbone, varying the structure and ring size of the cyclic peptide. SICLOPPS libraries have been combined with a bacterial reverse two-hybrid system in our labs and used in the identification of inhibitors of several protein–protein interactions. This protocol is expected to take around 3–4 weeks to implement.
- School of Chemistry, University of Southampton, Southampton SO17 1BJ, UK.
- Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Correspondence to: Stephen J Benkovic2 e-mail: sjb1@psu.edu
nature-products
A-Z product listing
- 0.2 cm gene pulser cuvettes(Bio-Rad Laboratories)
- 0.2% (w/v) arabinose solution(CAS)
- 10
thermophilic DNA polymerase buffer and Taq DNA polymerase(Promega) - Agarose, molecular biology grade(CAS)
- Appropriate 10 restriction enzyme buffer, 100 BSA and restriction enzymes(New England Biolabs)
- Chitin affinity resin(New England Biolabs)
