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Updated: Sep 28, 2026

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
Published on: March 25, 2020
Intein-mediated purification of cytotoxic endonuclease I-TevI by insertional inactivation and pH-controllable
Wei Wu1, David W Wood, Georges Belfort
1Wadsworth Center, New York State Department of Health and State University of New York at Albany, Albany, NY 12201-2002, USA.
Abstract:
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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