PROTEIN PURIFICATION USING INTEIN C-TERMINAL CLEAVAGE

本项目的目的是开发一种新的蛋白表达和纯化系统，该系统具有GST或MBP系统的优点，而没有额外的蛋白水解切割步骤的缺点。为了实现这一点，利用蛋白质剪接元件（称为内含肽），因为它们能够催化高度特异性的肽键切割反应。该系统的设计如下：将靶蛋白融合到修饰的内含肽的C-末端，其中插入几丁质结合结构域以允许亲和纯化，并且将MBP片段融合到内含肽的N-末端以允许有利的翻译起始。该项目的重点是研究温度、pH和/或巯基试剂对内含肽C-末端肽键断裂的诱导作用。将测试所有微型内含肽（缺乏内切核酸酶结构域的那些）改善蛋白质表达并允许融合蛋白在酵母中分泌的能力。从摇瓶转移到小型发酵罐，调查还将检查在高细胞密度发酵条件下的裂解和分泌控制。我们的目标是使这种内含肽介导的纯化系统立即适用于重组蛋白的大规模商业生产。拟议的商业应用：该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的纯化过程，并且可以立即适用于治疗性蛋白质的大规模商业生产。

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.