title: Traceless Production of Cyclic Peptide Libraries in E. coli.
abstract: Split intein circular ligation of peptides and proteins (SICLOPPS) is a genetically encoded method for the intracellular production of cyclic peptide libraries of around 100 million (10(8)) members that utilizes the Synechocystis sp PCC6803 (Ssp) DnaE split inteins. However, Ssp inteins are relatively slow splicing and intolerant of amino acid variation around the splice junction, potentially limiting the utility and composition of SICLOPPS libraries. In contrast, Nostoc punctiforme (Npu) DnaE split inteins not only splice significantly faster, they are also much more tolerant of amino acid variation around their splice junctions. Here, we report the use of engineered Npu inteins in SICLOPPS for the generation of cyclic peptide libraries and cyclic proteins. Despite their superior splicing characteristics, however, we observed a high level of toxicity from the Npu SICLOPPS constructs in E. coli. The observed toxicity was overcome though incorporation of an SsrA tag to target the spliced Npu inteins to the ClpXP complex for degradation. The resulting traceless Npu SICLOPPS inteins showed no toxicity to E. coli, demonstrating their potential for the production of cyclic peptide libraries for use in a variety of high-throughput screens.
authors.affiliation: Chemistry, University of Southampton , Southampton, SO17 1BJ, United Kingdom.
authors.forename: Jaime E
authors.initials: JE
authors.lastname: Townend
authors.affiliation: Chemistry, University of Southampton , Southampton, SO17 1BJ, United Kingdom.
authors.forename: Ali
authors.initials: A
authors.lastname: Tavassoli
mesh_terms: D001089:Arabinose / Q000378:metabolism; D049071:Endopeptidase Clp / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics; D047668:Inteins; D046937:Nostoc / Q000235:genetics / Q000378:metabolism; D019151:Peptide Library; D010456:Peptides, Cyclic / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids; D019154:Protein Splicing; D020798:Two-Hybrid System Techniques
chemical_list: D029968:Escherichia coli Proteins; D019151:Peptide Library; D010456:Peptides, Cyclic; C000622578:cyclo(cysteinyl-leucyl-leucyl-phenylalanyl-valyl-tyrosyl); D049452:Green Fluorescent Proteins; D001089:Arabinose; C483573:ClpXP protease, E coli; D049071:Endopeptidase Clp