Overproduction of Human Immunodeficiency Virus Type I Reverse Transcriptase in Escherichia coli and Purification of the Enzyme
Overproduction of Human Immunodeficiency Virus Type I Reverse Transcriptase in Escherichia coli and Purification of the Enzyme
复制标题
人类免疫缺陷病毒 I 型逆转录酶在大肠杆菌中的过量产生及酶的纯化
DOI：
--
复制
发表时间：
1990
影响因子：
2.6
通讯作者：
H. Shinagawa
中科院分区：
文献类型：
--
作者：
A. Saitoh;H. Iwasaki;A. Nakata;A. Adachi;H. Shinagawa
设计逆转录酶在E.杆菌为了高水平表达，HIV蛋白表达为与β-半乳糖苷酶的蛋白融合物。当覆盖gag基因3′端一半和整个pol基因的前病毒DNA片段连接到lacZ基因的3′端，使截短的gag与lacZ在读码框内融合时，产生少量逆转录酶，表明发生了移码和翻译后加工。当整个pol区域直接融合到lacZ基因时，产生了更多的逆转录酶。从1升细菌培养物中，获得1 mg高度纯化的逆转录酶，其由大约等摩尔量的两种物质（p64和p51）组成。这些蛋白质具有与推导的氨基酸序列一致的相同的N末端，因此，可以在E. pol区编码的蛋白酶对大肠杆菌的酶活性进行了研究。纯化的逆转录酶与病毒颗粒中纯化的酶具有相同的酶促活性，并与HIV携带者血清呈免疫反应性，具有较高的敏感性和特异性。
Overexpression of the reverse transcriptase was designed in E. coli. For a high level of expression, HIV protein was expressed as a protein fusion with β‐galactosidase. When the proviral DNA fragment covering the 3′ half of the gag gene and the entire pol gene was ligated to the 3′ end of the lacZ gene to fuse the truncated gag to lacZ in frame, a small quantity of reverse transcriptase was produced, indicating that frameshifting and post‐translational processing have occurred. Much more reverse transcriptase was produced when the entire pol region was directly fused to the lacZ gene. From a one liter culture of bacteria, 1 mg of highly purified reverse transcriptase consisting of approximately equimolar amounts of two species (p64 and p51) was obtained. These proteins had identical N‐termini consistent with the deduced amino acid sequence and therefore, might be correctly processed from the fusion protein in E. coli by the protease encoded by the pol region. The purified reverse transcriptase was enzymatically as active as the enzyme purified from the virus particles, and immunoreactive to the sera of HIV carriers with high sensitivity and specificity.
DOI：
10.1016/s0021-9258(18)61070-1
发表时间：
1987-07
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
P. Matsudaira
通讯作者：
P. Matsudaira