Subtilisin-like serine protease from hyperthermophilic archaeon Thermococcus kodakaraensis with N- and C-terminal propeptides
Subtilisin-like serine protease from hyperthermophilic archaeon Thermococcus kodakaraensis with N- and C-terminal propeptides
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DOI：
10.1093/protein/gzp092
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发表时间：
2010-05-01
影响因子：
2.4
通讯作者：
Kanaya, S.
中科院分区：
文献类型：
--
作者：
Foophow, T.;Tanaka, S.;Kanaya, S.
超嗜热古菌Thermococcus kodakaraensis的基因组含有编码枯草杆菌蛋白酶样丝氨酸蛋白酶的三个基因，Tk-1689、Tk-0076和Tk-枯草杆菌蛋白酶。其中，TK-枯草杆菌蛋白酶的结构和功能已被广泛研究。为了检测Tk-1689是否成熟为活性形式并作为Tk-枯草杆菌蛋白酶那样的超热稳定蛋白酶起作用，在大肠杆菌中过表达编码Tk-1689衍生物的基因，该基因不具有推定的N-末端信号序列，称为Pro-Tk-SP。Pro-Tk-SP由640个氨基酸残基组成，分子量为68.6 kDa。然而，重组蛋白被纯化为活性44 kDa蛋白酶，称为Tk-SP，其缺乏N-末端113和C-末端101个氨基酸残基。这一结果表明，Pro-Tk-SP由N端前肽（Ala 1-Ala 113）、成熟结构域（Tk-SP，Val 114-Val 539）和C端前肽（Asp 540-Gly 640）组成。与Tk-subtilisin一样，Tk-SP具有广泛的底物特异性和高度的热稳定性。其最适活性温度为100 ℃，其在100 ℃的半衰期为100分钟。它是完全耐用5%SDS，8 M尿素或10%Triton X-100处理。然而，与Tk-枯草杆菌蛋白酶和细菌枯草杆菌蛋白酶不同，Tk-SP既不需要Ca 2+也不需要前肽进行折叠。结果，Tk-SP即使在10 mM EDTA存在下也具有完全活性。因此，Tk-SP在耐热性、抗变性剂、抗洗涤剂和抗螯合剂等方面具有其他蛋白酶无法比拟的优势，在生物技术领域具有巨大的应用潜力。
The genome of the hyperthermophilic archaeon Thermococcus kodakaraensis contains three genes encoding subtilisin-like serine proteases, Tk-1689, Tk-0076 and Tk-subtilisin. Of them, the structure and function of Tk-subtilisin have been extensively studied. To examine whether Tk-1689 is matured to an active form and functions as a hyperthermostable protease as is Tk-subtilisin, the gene encoding the Tk-1689 derivative without a putative N-terminal signal sequence, termed Pro-Tk-SP, was overexpressed in Escherichia coli. Pro-Tk-SP is composed of 640 amino acid residues and its molecular mass is 68.6 kDa. The recombinant protein was purified, however, as an active 44 kDa protease, termed Tk-SP, which lacks the N-terminal 113 and C-terminal 101 amino acid residues. This result suggests that Pro-Tk-SP consists of an N-terminal propeptide (Ala1-Ala113), a mature domain (Tk-SP, Val114-Val539) and a C-terminal propeptide (Asp540-Gly640). Like Tk-subtilisin, Tk-SP showed a broad substrate specificity and was highly thermostable. Its optimum temperature for activity was similar to 100 degrees C and its half-life at 100 degrees C was 100 min. It was fully resistant to treatment with 5% SDS, 8 M urea or 10% Triton X-100. However, unlike Tk-subtilisin and bacterial subtilisins, Tk-SP requires neither Ca2+ nor propeptide for folding. As a result, Tk-SP was fully active even in the presence of 10 mM EDTA. Thus, Tk-SP has a great advantage over other proteases in high resistance to heat, denaturants, detergents and chelating agents and therefore has great potential for application in biotechnology fields.