PROTEIN PURIFICATION USING INTEIN C-TERMINAL CLEAVAGE

使用内含子 C 端切割纯化蛋白质

• 批准号: 6386918
• 负责人: Shaorong Chong
• 金额: $ 23.77万
• 依托单位: NEW ENGLAND BIOLABS, INC.
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6386918
• 关键词: affinity chromatography; chemical cleavage; molecular cloning; peptides; posttranslational modifications; protein engineering; protein purification; recombinant proteins; technology /technique development

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.

该项目的目的是开发一种新型的蛋白质表达和纯化系统，该系统具有GST或MBP系统的优势，而没有其他蛋白水解切割步骤的缺点。为了实现这一目标，蛋白质剪接元件（命名为inteins）用于催化高度特异性的肽键裂解反应。该系统的设计如下：靶蛋白融合到修饰的内部内部的C末端，其中插入了几丁质结合结构域以允许亲和力纯化，并将MBP片段融合到内端的N末端，以允许有利的翻译开始。该项目的重点是研究通过温度，pH和/或硫醇试剂在内部内部的C末端诱导肽键裂解。所有迷你intein（缺乏核酸内切酶结构域）将因其改善蛋白质表达并允许在酵母中分泌融合蛋白的能力而进行测试。从Shake-Flask移动到小型发酵罐，研究还将检查在高密度密度发酵条件下对裂解和分泌的控制。目的是使该内元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：这项研究导致了新型蛋白质表达和纯化系统的发展，该系统可能会显着简化重组蛋白的纯化过程，并可能立即适用于大规模商业生产治疗蛋白。