Intein-mediated rapid purification of Cre recombinase
Intein-mediated rapid purification of Cre recombinase
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DOI：
10.1006/prep.2001.1428
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发表时间：
2001-06-01
影响因子：
1.6
通讯作者：
Chong, SR
中科院分区：
文献类型：
--
作者：
Cantor, EJ;Chong, SR
由噬菌体pi产生的Cre重组酶催化原核和真核细胞中loxP识别位点之间的DNA的位点特异性重组，并且已广泛用于基因组工程和体外克隆。重组Cre已在大肠杆菌中过量生产，其纯化涉及多个步骤。在这份报告中，我们使用了一个“内含肽”融合系统来表达Cre作为一个C-末端融合到一个修饰的蛋白质剪接元件，即，内含肽，修饰的内含肽含有环状芽孢杆菌几丁质结合结构域，其允许将融合蛋白结合在几丁质柱上，并且可以被诱导进行体外肽键切割，其特异性地从柱中释放Cre。使用内含肽系统，我们在仅一个层析步骤之后获得了高纯度的非标记Cre，其对应于约80%的回收率和27倍的纯化。在体外测定系统中测定纯化的Cre的活性，发现其在超过6个月的时间内保持稳定。(C)北京：科学出版社.
Cre recombinase produced by bacteriophage pi catalyzes site-specific recombination of DNA between loxP recognition sites in both prokaryotic and eukaryotic cells and has been widely used for genome engineering and in vitro cloning. Recombinant Cre has been overproduced in Escherichia coli and its purification involves multiple steps. In this report, we used an "intein" fusion system to express Cre as a C-terminal fusion to a modified protein splicing element, i.e., intein, The modified intein contained a Bacillus circulans chitin-binding domain which allowed binding of the fusion protein on a chitin column and could be induced to undergo in vitro peptide bond cleavage which specifically released Cre from the column, Using the intein system, we have obtained highly pure non-tagged Cre after just a single chromatographic step, which corresponded to approximately 80% recovery and 27-fold purification. The activity of the purified Cre was determined in an in vitro assay system and was found to remain stable over a period of more than 6 months. (C) 2001 Academic Press.