期刊
NUCLEIC ACIDS RESEARCH
卷 30, 期 22, 页码 4864-4871出版社
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkf621
关键词
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资金
- NIGMS NIH HHS [R01 GM044844, R01 GM039422, GM44844, GM39422, R37 GM039422] Funding Source: Medline
An intein-mediated approach was developed for expression and affinity purification of a protein that is lethal to Escherichia coli. The protein, I-TevI, is an intron-encoded endonuclease. The approach involved the insertional inactivation of I-TevI with a controllable mini-intein placed in front of a cysteine required for splicing (an I-TevI::intein fusion). The purification was facilitated by a chitin-binding domain inserted into the mini-intein. Affinity purification of the I-TevI::intein fusion precursor on a chitin column was followed by pH-controllable splicing to restore the structure and function of I-TevI. To study the impact of the insertion context on I-TevI inactivation, the chimeric intein was inserted independently in front of seven cysteines of I-TevI. One of the seven intein integrants yielded I-TevI of high activity. This technique is, in principle, generalizable to the expression and purification of other cytotoxic proteins and is amenable to scale-up.
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