Affiliation:
1. New England Biolabs, Beverly, MA, USA
Abstract
The Mycobacterium xenopi gyrase A mini-intein has been engineered to yield a controllable N-terminal or C-terminal, single- splice-junction autocleavage element. When combined with an affinity tag, these modified mini-inteins can be used to purify target proteins after a single combined chromatography/cleavage step. Cleavage at the intein N terminus was induced with thiol reagents, while cleavage at the intein C terminus was induced by a temperature shift to 16°–25°C. Different preferences for the residue immediately preceding the intein were observed during thiol-induced, N-terminal splice-junction cleavage of the M. xenopi gyrase A mini-intein vs. the Saccharomyces cerevisiae vacuolar ATPase, subunit A (VMA) intein present in the IMPACTTM purification system. Furthermore, the M. xenopi gyrase A mini-intein Cterminal autocleavage vector allows isolation of polypeptides with N-terminal cysteine residues that are active in the Intein Mediated Protein Ligation method of protein semisynthesis.
Subject
General Biochemistry, Genetics and Molecular Biology,Biotechnology
Cited by
216 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献