Traceless Production of Cyclic Peptide Libraries in E. coli
Traceless Production of Cyclic Peptide Libraries in E. coli
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DOI：
10.1021/acschembio.6b00095
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发表时间：
2016-06-01
影响因子：
4
通讯作者：
Tavassoli, Ali
中科院分区：
文献类型：
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作者：
Townend, Jaime E.;Tavassoli, Ali
肽和蛋白质的断裂内含肽环状连接（SICLOPPS）是一种遗传编码的方法，用于利用集胞藻属物种PCC 6803（Ssp）DnaE断裂内含肽在细胞内产生约1亿（10（8））个成员的环状肽文库。然而，Ssp内含肽是相对缓慢的剪接并且不耐受剪接点周围的氨基酸变异，潜在地限制了SICLOPPS文库的实用性和组成。相比之下，念珠藻（Npu）DnaE分裂内含肽不仅剪接显着更快，他们也更容忍其剪接点周围的氨基酸变异。在这里，我们报告的用途：工程Npu内含肽在SICLOPPS的环状肽库和环状蛋白质的产生。尽管Npu SICLOPPS具有上级剪接特性，但我们观察到其在大肠杆菌中具有高水平的毒性。杆菌通过掺入SsrA标签以将剪接的Npu内含肽靶向ClpXP复合物进行降解来克服抑制的毒性。得到的无痕Npu SICLOPPS内含肽对大肠杆菌没有毒性。Tali，证明了它们用于生产用于各种高通量筛选的环肽文库的潜力。
Split intein circular ligation of peptides and proteins (SICLOPPS) is a genetically encoded, method for the intracellular production of cydic 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. tali, demonstrating their potential for the production of cyclic peptide libraries for -use in a variety of high -throughput screens.