PROTEIN PURIFICATION USING INTEIN C TERMINAL CLEAVAGE

描述：(改编自申请人的摘要)蛋白质剪接 涉及高度特异的自我催化的多肽键裂解反应 为蛋白质工程和蛋白质合成提供了新的途径。 结合酵母菌独特的自催化特性 用亲和层析法测定酿酒酵母VMA内蛋白(SCE VMA Intein)，a 新的蛋白质纯化方法已经被开发出来，它具有 极大地降低工业生产成本的潜力 酵素。利用硫代诱导的N-末端裂解活性 ，我们已经能够获得高度纯化的蛋白质 在单列之后。然而，融合蛋白的表达水平 蛋白质随目标蛋白质的不同而不同，这有时会导致 收益率非常低。此项目应通过以下方式解决此表达式问题 利用SCE VMA内含子的可诱导的C端切割活性。 目标蛋白将与内含素的C-末端融合 使融合蛋白的N-端序列得到优化 以获得高水平的蛋白质表达。这个C-末端切割系统 将首先在大肠杆菌中进行研究，然后应用于其他 具有分泌能力的异源蛋白表达系统，例如 毕赤亚·巴斯多里斯。后者可能会被自动化，适用于大型- 规模化工业生产。 建议的商业应用：不可用

DESCRIPTION: (Adapted from the applicant's abstract) Protein splicing involves highly specific, self-catalyzed peptide bond cleavage reactions which provide new avenue for protein engineering and protein synthesis. Combining the unique self-catalytic property of the Saccharomyces cerevisiae VMA intein (Sce VMA intein) with affinity chromatography, a novel protein purification methodology has been developed which has the potential to drastically reduce the production cost of industrial enzymes. Utilizing the thio-inducible-N-terminal cleavage activity of the Sce VMA intein, we have been able to obtain highly purified proteins after a single column. However, the expression level of the fusion protein varies with the target protein which can sometimes result in very low yield. This project should solve this expression problem by using the inducible C-terminal cleavage activity of the Sce VMA intein. The target protein will be fused to the C-terminus of the intein allowing the N-terminal sequence of the fusion protein to be optimized for high level of protein expression. This C-terminal cleavage system will first be studied in Escherichia coli and then applied to other heterologous protein expression systems with secretory capability, e.g. Pichia pastoris. The latter can potentially be automated for large- scale industrial productions. PROPOSED COMMERCIAL APPLICATION: NOT AVAILABLE