PROTEIN PURIFICATION USING INTEIN C-TERMINAL CLEAVAGE

本项目的目标是开发一种新型的蛋白质表达和纯化系统，该系统既具有GST或MBP系统的优点，又不存在额外的蛋白水解酶步骤的缺点。为了实现这一点，蛋白质剪接元件(称为内含素)被利用，因为它们能够催化高度特异的肽键断裂反应。该系统的设计如下：将目的蛋白融合到修饰的内含素的C端，其中插入甲壳素结合域以进行亲和纯化，并将MBP片段融合到内含素的N端以实现良好的翻译开始。本项目的重点是研究温度、pH和/或硫醇试剂对内含子C末端多肽键断裂的诱导作用。所有迷你内含子(那些缺乏核酸内切酶结构域的)都将接受测试，以确定它们是否有能力改善蛋白质表达，并允许在酵母中分泌融合蛋白质。从摇瓶到小型发酵罐，调查还将检查在高细胞密度发酵条件下对卵裂和分泌的控制。其目标是使这种由内含素介导的纯化系统立即适用于大规模商业生产重组蛋白。建议的商业应用：这项研究导致了一种新的蛋白质表达和纯化系统的开发，该系统可以显著简化重组蛋白质的纯化过程，并可能立即适用于大规模治疗性蛋白质的商业生产。

The objective of this project is to develop a novel protein expression and purification system which has the advantages of the GST or MBP systems without the drawbacks of additional proteolytic cleavage steps. To achieve this, protein splicing elements (named inteins) are utilized for their ability to catalyze highly specific peptide bond cleavage reactions. The design of the system is as follows: a target protein is fused to the C-terminus of a modified intein in which the chitin-binding domain is inserted to allow affinity purification and an MBP fragment is fused to the N-terminus of the intein to allow favorable translational start. The focus of the project is to investigate induction of peptide bond cleavage at the C-termini of the intein by temperature, pH and/or a thiol reagent. All mini-inteins (those that lack the endonuclease domain) will be tested for their ability to improve protein expression and allow secretion of the fusion protein in yeast. Moving from shake-flask to a small fermentor, the investigation will also examine control of cleavage and secretion under high-cell-density fermentation conditions. The goal is to make this intein-mediated purification system immediately applicable to large-scale commercial production of recombinant proteins. PROPOSED COMMERCIAL APPLICATIONS: This research leads to the development of a novel protein expression and purification system which may significantly simplify the purification process of recombinant proteins and may be immediately applicable to large-scale commercial production of therapeutic proteins.