Wu Wei, Wood David W, Belfort Georges, Derbyshire Victoria, Belfort Marlene
Wadsworth Center, New York State Department of Health and State University of New York at Albany, Albany, NY 12201-2002, USA.
Nucleic Acids Res. 2002 Nov 15;30(22):4864-71. doi: 10.1093/nar/gkf621.
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.
开发了一种内含肽介导的方法，用于表达和亲和纯化对大肠杆菌具有致死性的蛋白质。该蛋白质I-TevI是一种内含子编码的内切核酸酶。该方法包括用置于剪接所需半胱氨酸前的可控小型内含肽对I-TevI进行插入失活（I-TevI::内含肽融合体）。通过插入到小型内含肽中的几丁质结合结构域促进纯化。在几丁质柱上对I-TevI::内含肽融合前体进行亲和纯化，随后进行pH可控剪接以恢复I-TevI的结构和功能。为了研究插入背景对I-TevI失活情况的影响，将嵌合内含肽独立插入I-TevI的七个半胱氨酸前。七个内含肽整合体之一产生了高活性的I-TevI。该技术原则上可推广用于其他细胞毒性蛋白质的表达和纯化，并且适合扩大规模。
