Tavassoli Ali, Benkovic Stephen J
School of Chemistry, University of Southampton, Southampton SO17 1BJ, UK.
Nat Protoc. 2007;2(5):1126-33. doi: 10.1038/nprot.2007.152.
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 10(8) 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.
化学生物学的最新进展以及体内筛选所具有的优势凸显了构建一个强大且灵活的生物合成小分子文库的必要性。在此，我们描述了一种利用肽与蛋白质的分裂内含肽环化连接法（SICLOPPS）在大肠杆菌中生物合成多达10⁸个成员的环肽文库的方法。该方法利用分裂内含肽化学对随机化的肽序列进行环化。环肽文库的大小理论上可以是任意的，且肽本身可能包含无限制数量的随机残基。然而，文库大小受大肠杆菌转化效率的限制，随机残基一般限制为五个，但环肽主链中可使用额外的氨基酸，从而改变环肽的结构和环大小。在我们实验室中，SICLOPPS文库已与细菌反向双杂交系统相结合，并用于鉴定几种蛋白质 - 蛋白质相互作用的抑制剂。预计该方案实施大约需要3至4周时间。
