Intein-mediated purification of cytotoxic endonuclease I-TevI by insertional inactivation and pH-controllable splicing.
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.
Wu
Wei
W
Wadsworth Center, New York State Department of Health and State University of New York at Albany, Albany, NY 12201-2002, USA.
Wood
David W
DW
Belfort
Georges
G
Derbyshire
Victoria
V
Belfort
Marlene
M
D002846:Chromatography, Affinity; D003545:Cysteine / Q000235:genetics; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000633:toxicity; D004926:Escherichia coli / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D008954:Models, Biological; D008958:Models, Molecular; D016254:Mutagenesis, Insertional / Q000379:methods*; D019154:Protein Splicing
D004706:Endodeoxyribonucleases; C065074:I-TEVI endonuclease; D003545:Cysteine