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Traceless production of cyclic peptide libraries in E. coli

Traceless production of cyclic peptide libraries in E. coli
Traceless production of cyclic peptide libraries in E. coli
Split intein circular ligation of peptides and proteins (SICLOPPS) is a genetically encoded method for the intracellular production of cyclic peptide libraries of around a hundred million (108) members that utilizes the Synechocystis sp PCC6803 (Ssp) DnaE split inteins. However, Ssp inteins are relatively slow splicing and intolerant of amino acids variation around the splice junction potentially limiting the utility and composition of SICLOPPS libraries. In contrast, Noctus 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.
1554-8929
1624-1630
Townend, Jaime
598d5099-60ba-4a5b-89dc-b4c674c1aeda
Tavassoli, Ali
d561cf8f-2669-46b5-b6e1-2016c85d63b2
Townend, Jaime
598d5099-60ba-4a5b-89dc-b4c674c1aeda
Tavassoli, Ali
d561cf8f-2669-46b5-b6e1-2016c85d63b2

Townend, Jaime and Tavassoli, Ali (2016) Traceless production of cyclic peptide libraries in E. coli. ACS Chemical Biology, 11, 1624-1630. (doi:10.1021/acschembio.6b00095). (PMID:27027149)

Record type: Article

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 a hundred million (108) members that utilizes the Synechocystis sp PCC6803 (Ssp) DnaE split inteins. However, Ssp inteins are relatively slow splicing and intolerant of amino acids variation around the splice junction potentially limiting the utility and composition of SICLOPPS libraries. In contrast, Noctus 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.

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Accepted/In Press date: 30 March 2016
e-pub ahead of print date: 30 March 2016

Identifiers

Local EPrints ID: 390752
URI: http://eprints.soton.ac.uk/id/eprint/390752
ISSN: 1554-8929
PURE UUID: 0f7076ea-1bc8-45e2-9806-60239de69d30
ORCID for Ali Tavassoli: ORCID iD orcid.org/0000-0002-7420-5063

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Date deposited: 07 Apr 2016 08:55
Last modified: 18 Feb 2021 17:07

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