摘要
基于蛋白质内含子的蛋白质纯化自我断裂标签已经被广泛使用超过15年之久.但这一系统体内表达过程的提前断裂一直是限制这一技术广泛应用的瓶颈,特别是在需要高温表达和长表达周期的真核表达系统中.本研究介绍了一种利用小肽控制的基于蛋白质内含子和非层析标签ELP(elastin-like polypeptide)的自我断裂系统.在这一系统中,蛋白质内含子的体内外活性严格受到其结构互补小肽控制.在体内表达不含有互补小肽时,蛋白质内含子不具有活性;而在体外添加结构互补小肽,蛋白质内含子结构恢复并发生C端断裂反应释放目的蛋白.由于非层析标签ELP的引入,因此整个纯化过程可以简单地通过几步机械沉淀完成.此外,这一系统反应pH、小肽与前体蛋白之间的摩尔比及断裂速率也一并进行了系统的研究.
Intein-based self-cleaving tags have been widely applied in protein purification for over fifteen years. However, premature cleavage in vivo during expression remains a major concern for this technique, particularly in eukaryotic host systems that have long expression periods. We developed a peptide triggered self-cleaving tag based on an intein and ELP （elastin-like polypeptide） tag. In this system, the intein activity was tightly controlled by the addition of a compatible peptide. During expression in vivo, the intein had no activity without the compatible peptide, while the intein activity could be restored in vitro with the complementary peptide. Through the non-chromatographic ELP tag, the whole purification could be easily performed by several steps of mechanical precipitation. The cleaving conditions, including pH, the ratio between the peptide and the precursor and the cleaving kinetics were also studied here.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2014年第2期202-208,共7页
Chinese Journal of Biochemistry and Molecular Biology
基金
Supported by National Natural Science Foundation ofChina(No.31070698)
Shanghai Key Projects of Basic Research(No.10JC1400300)
Innovation Foundation of Donghua University for PhD Candidates(No.12D11320)~~
