ABSTRACT

A system based on cyanobacterial split inteins, SICLOPPs (Split Intein Circular Ligation of Proteins and Peptides), has been used to synthesise a small natively cyclic plant protein, kalata B1, and cyclised versions of the natively linear therapeutic peptides ziconotide and leconotide. The cyclic versions of these naturally linear peptides include linker sequences between their native termini to allow the correct tertiary structure to form. The native structure of each includes three disulphide bonds characteristic of knottins. Cyclic permutations of leconotide yielded different proportions of correctly spliced product, identifying an optimal splice site and revealing the influence of residues around the splice junction. The rate of splicing was manipulated to facilitate affinity purification prior to intein-mediated removal of the affinity tag.

摘要

基于蓝细菌分裂内含蛋白，SICLOPPs（蛋白质和肽的内含拆分环连接）的系统已用于合成小的天然环状植物蛋白，kalata B1以及天然线性治疗性肽Ziconotide和Leconotide的环化形式。这些天然线性肽的环状形式在其天然末端之间包含接头序列，以形成正确的三级结构。每一个的天然结构均包含结蛋白特征性的三个二硫键。莱考诺肽的循环排列产生了不同比例的正确剪接的产物，从而确定了最佳的剪接位点并揭示了剪接点周围残基的影响。在内含肽介导的亲和标签去除之前，控制剪接速率以促进亲和纯化。