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

摘要

An intein-mediated approach was developed for expression and affinity purification of a protein that is lethal to Escherichia coli. The protein, I-Tevl, is an intron-encoded endonuclease. The approach involved the insertional inactivation of I-Tevl with a controllable mini-intein placed in front of a cysteine required fro splicing (an I-Tevl::intein fusion). The purification was facilitated by a chitin-binding domain inserted into the mini-intein. Affinity purification of the I-Tevl::intein fusion precursor on a chitin column was followed by pH-controllable splicing to restore the structure and function of I-Tevl. To study the impact of the insertion context on I-Tevl inactivation, the chimeric intein was inserted independently in front of seven cysteines of I-Tevl. One of the seven intein integrants yielded I-Tevl of high activity. This technique is, in principle, generalizable to the expression and purification of other cytotoxic proteins and is amenable to scale-up.