Cloning and expression of an intracellular alkaline protease gene from alkalophilic Thermoactinomyces sp. HS682.
Cloning and expression of an intracellular alkaline protease gene from alkalophilic Thermoactinomyces sp. HS682.
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嗜碱嗜热放线菌胞内碱性蛋白酶基因的克隆和表达。
DOI：
10.1271/bbb.61.298
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Tetsu Kimura
中科院分区：
文献类型：
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作者：
K. Tsuchiya;Ichiro Ikeda;Takashi Tsuchiya;Tetsu Kimura
克隆嗜碱高温放线菌HS 682胞内碱性丝氨酸蛋白酶基因，并在大肠杆菌中表达。序列分析表明，该基因包含一个启动子区、一个转录终止信号区和一个963个碱基的开放阅读框，编码321个氨基酸的多肽。在E. coli经DEAE-Toyopolysin 650 M和Sephadex G-75柱层析纯化。纯化蛋白的N-末端序列（30个氨基酸）与ORF推导的氨基酸序列Asp 16-Val 45一致。在纯化过程中去除了N-末端区域中的15个氨基酸。推导的氨基酸序列与微生物胞内丝氨酸蛋白酶具有高度的相似性。通过SDS-PAGE估计该酶的分子量为38 kDa。该酶在pH 6.0-12.0和60 ℃以下的Ca 2+存在下稳定。该酶的最适温度和pH分别为65 ℃和pH 11.0。DFP和PMSF对该酶有抑制作用，而MIA和EDTA对该酶无抑制作用。
An intracellular alkaline serine protease gene of alkalophilic Thermoactinomyces sp. HS682 was cloned and expressed in Escherichia coli. Sequence analysis showed a putative promoter region, a putative transcriptional termination signal, and an open reading frame of 963 bases, coding for a polypeptide of 321 amino acids. The protease expressed in E. coli was purified by DEAE-Toyopearl 650M and Sephadex G-75 chromatography. The N-terminal sequence (30 amino acids) of the purified protein was coincident with Asp16-Val45 of the deduced amino acid sequence of the ORF. Fifteen amino acids in the N-terminal region were removed during the purification procedures. The deduced amino acid sequence showed high similarity with microbial intracellular serine proteases. The molecular mass of this enzyme was estimated to be 38 kDa by SDS-PAGE. The enzyme was stable at pH 6.0-12.0 and below 60 degrees C in the presence of Ca2+. The temperature and pH optima of the enzyme were 65 degrees C and pH 11.0, respectively. The enzyme was inhibited by DFP and PMSF, but not by MIA and EDTA.