PROTEIN PURIFICATION USING INTEIN C TERMINAL CLEAVAGE

使用内含肽 C 末端切割纯化蛋白质

• 批准号: 2648078
• 负责人: Shaorong Chong
• 金额: $ 9.39万
• 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 1998-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2648078
• 关键词: affinity chromatography; chemical cleavage; molecular cloning; posttranslational modifications; protein engineering; protein purification

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

描述：（改编自申请人的摘要）蛋白质剪接 涉及高度特异性，自催化的肽键裂解反应 为蛋白质工程和蛋白质合成提供了新的途径。 结合糖疗法的独特自我催化特性 酿酒酵母VMA内蛋白（SCE VMA内无素），具有亲和色谱，A 已经开发了新型蛋白质纯化方法论 大幅降低工业生产成本的潜力 酶。 利用可诱导的N末端裂解活性 SCE VMA内蛋白，我们能够获得高度纯化的蛋白 单列之后。但是，融合的表达水平 蛋白质随靶蛋白而变化，有时会导致 产量非常低。 这个项目应该通过 使用SCE VMA内素的诱导型C末端裂解活性。 靶蛋白将融合到内因的C末端 允许优化融合蛋白的N末端序列 对于高水平的蛋白质表达。 该C末端裂解系统 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 Pichia Pastoris。 后者有可能自动化 规模工业生产。 拟议的商业应用程序：不可用

项目成果

Productive interaction of chaperones with substrate protein domains allows correct folding of the downstream GFP domain.

伴侣与底物蛋白结构域的有效相互作用允许下游 GFP 结构域的正确折叠。

• DOI: 10.1016/j.gene.2005.01.019
• 发表时间: 2005
• 期刊: Gene.
• 作者: Zhang,Aihua;Cantor,EricJ;Barshevsky,Tanya;Chong,Shaorong
• 通讯作者: Chong,Shaorong

Simulation of large-scale production of a soluble recombinant protein expressed in Escherichia coli using an intein-mediated purification system.

使用内含肽介导的纯化系统模拟在大肠杆菌中表达的可溶性重组蛋白的大规模生产。

• DOI: 10.1385/abab:126:2:093
• 发表时间: 2005
• 期刊: Applied biochemistry and biotechnology
• 影响因子: 3
• 作者: Sharma,ShamikS;Chong,Shaorong;Harcum,SarahW
• 通讯作者: Harcum,SarahW