Sakamoto S, Terada I, Iijima M, Ohta T, Matsuzawa H
Life Science Research Laboratory, Japan Tobacco Inc., Yokohama.
Biosci Biotechnol Biochem. 1995 Aug;59(8):1438-43. doi: 10.1271/bbb.59.1438.
The thermophilic protease aqualysin I (AQI) gene (aquI), derived from Thermus aquaticus YT-1, was inserted under the control of the bacteriophage T7 promoter in an expression plasmid. The plasmid was introduced into two strains of E. coli JM109 (DE3), one carrying and one lacking an F' episome, which carries the lacIq gene. Upon cultivation the strain carrying an F' episome produced AQI as an insoluble fusion protein (74kDa) with the T7 gene 10 protein. This insoluble protein could not be processed into mature AQI by heat treatment and thus it had no proteolytic activity. On the other hand, when the strain lacking an F' episome was used as a host cell for aquI expression, non-induced, or leaky, expression occurred, and AQI was produced in a soluble form. This soluble protein could be processed into active AQI by heat treatment. Moreover, when a low concentration of IPTG (0.0125 mM) was added, the amount of active AQI was 2.7 times greater than that produced in a batch culture without induction.
源自嗜热水栖菌YT-1的嗜热蛋白酶嗜热水解素I（AQI）基因（aquI）被插入到一个表达质粒中，置于噬菌体T7启动子的控制之下。该质粒被导入两株大肠杆菌JM109（DE3）中，一株携带F'附加体，另一株不携带，F'附加体携带lacIq基因。培养时，携带F'附加体的菌株产生了一种与T7基因10蛋白形成的不溶性融合蛋白（74kDa）形式的AQI。这种不溶性蛋白经热处理后不能加工成成熟的AQI，因此没有蛋白水解活性。另一方面，当不携带F'附加体的菌株用作aquI表达的宿主细胞时，会发生非诱导型或渗漏型表达，并且AQI以可溶形式产生。这种可溶性蛋白经热处理后可加工成有活性的AQI。此外，当添加低浓度的异丙基-β-D-硫代半乳糖苷（IPTG，0.0125 mM）时，活性AQI的产量比未诱导的分批培养所产生的产量高2.7倍。
