PROTEIN PURIFICATION USING INTEIN C-TERMINAL CLEAVAGE

该项目的目标是开发一种新的蛋白质表达和纯化系统，该系统具有GST或MBP系统的优点，而没有额外的蛋白质水解裂解步骤的缺点。为了实现这一目标，利用蛋白质剪接元件（称为interins）催化高度特异性肽键裂解反应的能力。该系统的设计如下：将目标蛋白融合到修饰的肠蛋白的c端，其中插入几丁质结合结构域以进行亲和纯化，并将MBP片段融合到肠蛋白的n端以允许有利的翻译开始。该项目的重点是研究温度、pH值和/或硫醇试剂对蛋白c端肽键裂解的诱导作用。所有的迷你蛋白（那些缺乏核酸内切酶结构域的蛋白）都将被测试它们在酵母中改善蛋白表达和允许融合蛋白分泌的能力。从摇瓶到小型发酵罐，研究还将检查在高密度发酵条件下对卵裂和分泌的控制。目标是使这种内部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：本研究将导致一种新的蛋白质表达和纯化系统的发展，该系统可能会大大简化重组蛋白的纯化过程，并可能立即适用于治疗蛋白的大规模商业生产。

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.