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Nucleic Acids Research, 2002, Vol. 30, No. 22 4864-4871
© 2002 Oxford University Press

Intein-mediated purification of cytotoxic endonuclease I-TevI by insertional inactivation and pH-controllable splicing

Wei Wu1, David W. Wood1,2, Georges Belfort2, Victoria Derbyshire1 and Marlene Belfort*,1

1 Wadsworth Center, New York State Department of Health and State University of New York at Albany, Albany, NY 12201-2002, USA and 2 Howard P. Isermann Department of Chemical Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA

*To whom correspondence should be addressed. Tel: +1 518 473 3345; Fax: +1 518 474 3181; Email: belfort{at}wadsworth.org
Present address:
David W. Wood, Department of Chemical Engineering, Princeton University, Princeton, NJ 08544, USA

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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