Intein-mediated purification of cytotoxic endonuclease I-TevI by insertional inactivation and pH-controllable splicing |
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| Authors: | Wu Wei Wood David W Belfort Georges Derbyshire Victoria Belfort Marlene |
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| Affiliation: | Wadsworth Center, New York State Department of Health and State University of New York at Albany, Albany, NY 12201-2002, USA. |
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| 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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