Functional expression and enzymatic properties of two Sitophilus zeamais cysteine proteinases showing different autolytic processing profiles in vitro.
Functional expression and enzymatic properties of two Sitophilus zeamais cysteine proteinases showing different autolytic processing profiles in vitro.
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两种玉米半胱氨酸蛋白酶的功能表达和酶学特性在体外表现出不同的自溶加工特性。
DOI：
10.1093/oxfordjournals.jbchem.a021993
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发表时间：
1998
影响因子：
2.7
通讯作者：
Y. Emori
中科院分区：
文献类型：
--
作者：
I. Matsumoto;K. Abe;S. Arai;Y. Emori
为了更详细地表征在我们以前的研究中克隆的来自玉米象（Sitophilus zeamais）的组织蛋白酶L样半胱氨酸蛋白酶（SCP）[松本等人（1997）生物化学杂志（J.Biochem.121），464-476]，我们建立了使用来自大肠杆菌的谷胱甘肽S-转移酶（GST）融合基因载体进行其功能表达和纯化的系统。将两种代表性SCP的酶原形式proSCPc 1和proSCPg 3表达为GST融合蛋白，并在谷胱甘肽琼脂糖柱上纯化。GST-proSCPc 1在酸性pH下有效地进行自身蛋白水解切割成成熟形式，并对包括血红蛋白和Z-Phe-Arg-MCA在内的各种底物表现出显著的蛋白水解活性。SCPc 1的活化形式的酶特性类似于哺乳动物组织蛋白酶L的酶特性，但其水解血红蛋白的最佳pH值明显较低。另一个proSCP，GST-proSCPg 3，其具有比SCPc 1更短的COOH-末端结构域，几乎不经历自溶加工，并且仅显示非常轻微的蛋白水解活性，尽管GST-proSCPg 3的其他酶特性与GST-proSCPc 1的那些相似。
To characterize in more detail the cathepsin L-like cysteine proteinases from Sitophilus zeamais (SCPs) cloned in our previous study [Matsumoto et al. (1997) J. Biochem. 121, 464-476], we established a system for their functional expression and purification using a glutathione S-transferase (GST) fusion gene vector from Escherichia coli. The proenzyme forms of two representative SCPs, proSCPc1 and proSCPg3, were expressed as GST-fusion proteins and purified on a glutathione Sepharose column. GST-proSCPc1 undergoes autoproteolytic cleavage into the mature form efficiently at acidic pH, and exhibits significant proteolytic activity toward various substrates including hemoglobin and Z-Phe-Arg-MCA. The enzymatic characteristics of the activated form of SCPc1 are similar to those of mammalian cathepsin L, but its pH optimum for the hydrolysis of hemoglobin is significantly lower. The other proSCP, GST-proSCPg3, which has a shorter COOH-terminal domain than SCPc1, undergoes almost no autolytic processing and shows only very slight proteolytic activity, although the other enzymatic characteristics of GST-proSCPg3 are similar to those of GST-proSCPc1.