TaqI限制性内切酶的过量生产、纯化及结晶

Overproduction, purification and crystallization of TaqI restriction endonuclease.

作者信息

Barany F

机构信息

Department of Microbiology, Hearst Microbiology Research Center, Cornell University Medical College, New York, NY 10021.

出版信息

Gene. 1988 May 30;65(2):167-77. doi: 10.1016/0378-1119(88)90453-2.

DOI:10.1016/0378-1119(88)90453-2

PMID:2842231

Abstract

Under phoA promoter control, TaqI endonuclease was overproduced to 5% of Escherichia coli cellular proteins. This was achieved by fusing the endonuclease gene to the first four codons of the alkaline phosphatase signal sequence. For maximal overproduction (30% of cellular proteins), a putative 14-bp hairpin within the endonuclease coding sequence was replaced with degenerate codons. In addition, TaqI methylase was required to protect host DNA. The endonuclease was purified in sufficient amounts for crystallization.

摘要

在phoA启动子的控制下，TaqI核酸内切酶的产量过量至大肠杆菌细胞蛋白质的5%。这是通过将核酸内切酶基因与碱性磷酸酶信号序列的前四个密码子融合来实现的。为了实现最大过量表达（细胞蛋白质的30%），核酸内切酶编码序列中的一个推定的14碱基对发夹结构被简并密码子取代。此外，需要TaqI甲基化酶来保护宿主DNA。纯化得到了足够用于结晶的核酸内切酶。