Skip Navigation

This Article
Right arrow Full Text Freely available
Right arrow Print PDF (218K) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (17)
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Wu, W.
Right arrow Articles by Belfort, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wu, W.
Right arrow Articles by Belfort, M.
Related Collections
Right arrow Recombinant DNA expression
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

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.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Nucleic Acids ResHome page
J. B. Robbins, M. Stapleton, M. J. Stanger, D. Smith, J. T. Dansereau, V. Derbyshire, and M. Belfort
Homing endonuclease I-TevIII: dimerization as a means to a double-strand break
Nucleic Acids Res., March 12, 2007; 35(5): 1589 - 1600.
[Abstract] [Full Text] [PDF]


Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.