Ono Toshio, Nemoto Takayuki K, Shimoyama Yu, Kimura Shigenobu, Ohara-Nemoto Yuko
Department of Oral Molecular Biology, Course of Medical and Dental Sciences, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8588, Japan.
Anal Biochem. 2008 Oct 1;381(1):74-80. doi: 10.1016/j.ab.2008.06.022. Epub 2008 Jun 20.
V8 protease (GluV8), a member of the glutamyl endopeptidase I family isolated from the V8 strain of Staphylococcus aureus, is widely used for proteome analysis because of its unique substrate specificity and resistance to detergents. We recently developed an Escherichia coli expression system for the production of GluV8 based on a technique that suppresses the autoproteolysis--the use of the prosequence of its homologue (GluSE) from Staphylococcus epidermidis as a chimeric form or the introduction of four substitutions in the prosequence of GluV8. In the current study, we refined this technique through five amino acid substitutions within the prosequence of GluV8 for complete suppression of the autodegradation. As a result, the recovery of GluV8 proform was enhanced to 20 fg/cell, which was comparable to the level of a constitutive inactive form of GluV8, indicating complete suppression of the autoproteolysis. This mutated propeptide was also effective for the expression of the mature sequence of the glutamyl endopeptidase from Staphylococcus warneri. The recombinant proteins were successfully converted to their active forms through a common cleavage mechanism mediated by thermolysin in vitro. This strategy may shed light on the way for the expression of the proteases that have been scarcely produced in E. coli to date.
V8蛋白酶（GluV8）是从金黄色葡萄球菌V8菌株中分离出来的谷氨酰胺内肽酶I家族的成员，由于其独特的底物特异性和对去污剂的抗性，被广泛用于蛋白质组分析。我们最近基于一种抑制自身蛋白水解的技术开发了一种用于生产GluV8的大肠杆菌表达系统——使用来自表皮葡萄球菌的同源物（GluSE）的前导序列作为嵌合形式，或者在GluV8的前导序列中引入四个取代。在本研究中，我们通过在GluV8的前导序列内进行五个氨基酸取代来完善该技术，以完全抑制自降解。结果，GluV8前体形式的回收率提高到了20 fg/细胞，这与GluV8组成型无活性形式的水平相当，表明自蛋白水解被完全抑制。这种突变的前肽对沃氏葡萄球菌谷氨酰胺内肽酶成熟序列的表达也有效。重组蛋白通过嗜热菌蛋白酶在体外介导的共同切割机制成功转化为其活性形式。该策略可能为迄今为止在大肠杆菌中很少产生的蛋白酶的表达提供思路。
