The in vitro ligation of bacterially expressed proteins using an intein from Methanobacterium thermoautotrophicum
The in vitro ligation of bacterially expressed proteins using an intein from Methanobacterium thermoautotrophicum
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DOI：
10.1074/jbc.274.7.3923
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发表时间：
1999-02-12
影响因子：
4.8
通讯作者：
Xu, MQ
中科院分区：
文献类型：
--
作者：
Evans, TC;Benner, J;Xu, MQ
已知的最小的内含肽，发现在嗜热甲烷杆菌的核糖核苷二磷酸还原酶基因（Mth RIR 1内含肽），被发现在大肠杆菌中剪接不良与天然存在的脯氨酸残基相邻的N-末端半胱氨酸的内含肽。当该脯氨酸被丙氨酸残基取代时，剪接效率增加。然而，通过分别用丙氨酸取代内含肽的C-末端天冬酰胺或N-末端半胱氨酸，产生了显示有效的N-末端和C-末端切割的构建体。此外，这些构建体用于在蛋白质序列上特异性地产生互补反应基团以用于连接反应。内含肽产生的C-末端硫酯与E.大肠杆菌麦芽糖结合蛋白（43 kDa）和T4 DNA连接酶（56 kDa）或硫氧还蛋白（12 kDa）的N末端处的内含肽产生的半胱氨酸导致蛋白质通过天然肽键连接。因此，最小的已知内含肽能够剪接，并且其独特的性质将内含肽介导的蛋白质连接的效用扩展到包括大的细菌表达的蛋白质的体外融合。
The smallest known intein, found in the ribonucleoside diphosphate reductase gene of Methanobacterium thermoautotrophicum (Mth RIR1 intein), was found to splice poorly in Escherichia coli with the naturally occurring proline residue adjacent to the N-terminal cysteine of the intein. Splicing proficiency increased when this proline was replaced with an alanine residue, However, constructs that displayed efficient N- and C-terminal cleavage were created by replacing either the C-terminal asparagine or N-terminal cysteine of the intein, respectively, with an alanine. Furthermore, these constructs were used to specifically generate complementary reactive groups on protein sequences for use in ligation reactions. Reaction between an intein-generated C-terminal thioester on E. coli maltose-binding protein (43 kDa) and an intein-generated cysteine at the N terminus of either T4 DNA ligase (56 kDa) or thioredoxin (12 kDa) resulted in the ligation of the proteins through a native peptide bond. Thus the smallest of the known inteins is capable of splicing and its unique properties extend the utility of intein-mediated protein ligation to include the in vitro fusion of large, bacterially expressed proteins.