The protease and reverse transcriptase of the tobacco LTR retrotransposon Tnt1 are enzymatically active when expressed in Escherichia coli
The protease and reverse transcriptase of the tobacco LTR retrotransposon Tnt1 are enzymatically active when expressed in Escherichia coli
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DOI：
10.1023/a:1010614918763
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发表时间：
2001-07-01
影响因子：
5.1
通讯作者：
Lucas, H
中科院分区：
文献类型：
--
作者：
Feuerbach, F;Lucas, H
将烟草反转录转座子Tnt1-94的开放阅读框(ORF)在大肠杆菌中高效表达，以检测其蛋白水解酶和逆转录酶活性。在大肠杆菌中，TNT1-94多聚蛋白被元件编码的蛋白酶切割，释放出表观分子质量为37 kDa的Gag蛋白，形成高密度聚集体。缺失分析确定的TNT1-94蛋白水解酶(D-T-A)的催化部位不同于逆转录病毒和已知的反转录转座子(D-T/S-G)。TNT1-94裂解或未裂解的ORF显示外源RT活性。植物反转录转座子ORF在大肠杆菌中的过表达为检测其蛋白质的酶活性和确定其催化部位提供了一种非常有用的策略。
The open reading frame (ORF) of the tobacco retrotransposon Tnt1-94 was over-expressed in Escherichia coli to assay its protease and reverse transcriptase (RT) enzymatic activities. In E. coli, Tnt1-94 polyprotein is cleaved off by the element-encoded protease to release a Gag protein with an apparent molecular mass of 37 kDa that forms high-density aggregates. The catalytic site of Tnt1-94 protease (D-T-A) as determined by deletion analysis differs from that of retroviruses and of well-characterized retrotransposons (D-T/S-G). The cleaved or uncleaved ORF of Tnt1-94 displays an exogenous RT activity. Over-expression of plant retrotransposons ORFs in E. coli provides a very useful strategy to assay the enzymatic activities of their proteins and to determine their catalytic sites.