高剪接活性断裂蛋白质内含子的体内切割  

Cleavage of Split-Inteins with High Splicing Activity

作　　者：林瑛[1] 周倩[1] 荀启静 孟清[1] 

机构地区：[1]东华大学生物科学与技术研究所,上海201620

出　　处：《中国生物工程杂志》2011年第8期97-101,共5页China Biotechnology

基　　金：国家自然科学基金(30800186;31070698);国家"863"计划(2006AA03Z451);教育部高等学校博士学科点专项科研基金(200802551026)资助项目

摘　　要：蛋白质内含子介导的断裂(切割)反应被用于蛋白质纯化、连接和环化等,但目前仍存在断裂效率低、断裂反应的不可控、产物复杂等问题。蛋白质内含子的定点突变可导致其N端或C端断裂。其末位氨基酸突变则剪接反应第3步天冬酰胺环化无法进行,发生N端断裂;其首位氨基酸发生突变则剪接反应第一步酰基重排及其后续步骤均无法进行,而天冬酰胺环化仍可进行,发生C端断裂。利用已获得的高剪接活性的S1和S11型断裂蛋白质内含子Ssp GyrB,分别将其参与剪接反应的首位半胱氨酸或末位天冬酰胺突变为丙氨酸,构建能够发生一端断裂的断裂蛋白质内含子。研究结果表明,突变后断裂蛋白质内含子的剪接反应几乎不发生,其断裂活性有不同程度的提高,获得了在大肠杆菌体内具有较高效断裂活性的断裂蛋白质内含子。这将为进一步研究其体外可控性剪接、构建高效的蛋白纯化系统和深入研究蛋白质内含子的剪接机制提供基础。Intein-based protein cleavage can be useful tools of recombinant protein purification,ligation and cyclization.However,existing methods were often complicated by low cleavage efficiency,spontaneous cleavages,and low yield of desired protein products.Site-specific cleavages at the N-or C-terminus of an intein can result from alterations of the protein splicing reaction.In N-terminal cleavage,the intein’s last residue is mutated to abolish step 3 of the splicing mechanism.Step 1 of the splicing mechanism can still happen,and the resulting ester bond can spontaneously hydrolyze to separate the N-extein from the intein.In C-cleavage,the intein’s first residue is mutated to abolish step 1 of the splicing mechanism,but Asn cyclization can still occur to break the peptide bond between the C-extein and intein.The first residue（Cys）or last residue（Asn）mutated split-inteins through site-specific mutagenesis using high splicing S1 and S11 Ssp GyrB split-inteins were constructed and their cleavage efficiency in E.coli cells were tested.Splicing was blocked by site-specific mutation,N-cleavage of S11 split-intein and C-cleavage of S1 split-intein were increased in E.coli cells.This will help us to investigate controllable protein cleavage in vitro,further establish efficient protein purification system and investigate splicing mechanism of split-inteins.

关 键 词：蛋白质内含子 断裂蛋白质内含子 定点突变 

分 类 号：Q71[生物学—分子生物学]