Specificity of the medaka enteropeptidase serine protease and its usefulness as a biotechnological tool for fusion-protein cleavage
Specificity of the medaka enteropeptidase serine protease and its usefulness as a biotechnological tool for fusion-protein cleavage
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DOI：
10.1073/pnas.0610447104
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发表时间：
2007-04-24
影响因子：
11.1
通讯作者：
Takahashi, Takayuki
中科院分区：
文献类型：
--
作者：
Ogiwara, Katsueki;Takahashi, Takayuki
我们克隆了两个不同的cDNA肠肽酶（EP）从青鳉，青鳉，这是一种小型淡水硬骨鱼的肠道。mRNA编码EP-1（1，036个残基）和EP-2（1，043个残基），两者都具有独特、保守的N末端重链和C末端催化丝氨酸蛋白酶轻链结构域结构。当与哺乳动物EP丝氨酸蛋白酶相比时，meclaka酶对小的合成肽底物表现出极低的酰胺分解活性。通过定点诱变产生青鳉EP蛋白酶的12种突变形式。其中，突变蛋白酶E173 A对合成底物和蛋白质底物的非特异性水解活性显著降低，而Asp-Asp-Asp-Asp-Lys（DA-切割活性）没有明显降低。对于含有D4 K-切割位点的融合蛋白的切割，青鳉EP蛋白酶显示出优于其哺乳动物对应物的优势。基于我们目前的数据，我们提出E173 A突变体是最合适的蛋白酶，特异性切割蛋白质含有D4 K切割序列。
We cloned two distinct cDNAs for enteropeptidase (EP) from the intestine of the medaka, Oryzias latipes, which is a small freshwater teleost. The mRNAs code for EP-1 (1,036 residues) and EP-2 (1,043 residues), both of which have a unique, conserved domain structure of the N-terminal heavy chain and C-terminal catalytic serine protease light chain. When compared with mammalian EP serine proteases, the meclaka enzyme exhibited extremely low amidolytic activity for small synthetic peptide substrates. Twelve mutated forms of the medaka EP protease were produced by site-directed mutagenesis. Among them, one mutant protease, E173A, was found to have considerably reduced nonspecific hydrolytic activities both for synthetic and protein substrates without serious reduction of its Asp-Asp-Asp-Asp-Lys (DA-cleavage activity. For the cleavage of fusion proteins containing a D4K-cleavage site, the medaka EP proteases were shown to have advantages over their mammalian counterparts. Based on our present data, we propose that the E173A mutant is the most appropriate protease to specifically cleave proteins containing the D4K cleavage sequence.