Importance of terminal residues on circularly permutated Escherichia coli alkaline phosphatase with high specific activity
Importance of terminal residues on circularly permutated Escherichia coli alkaline phosphatase with high specific activity
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DOI：
10.1263/jbb.100.197
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发表时间：
2005-08-01
影响因子：
2.8
通讯作者：
Ueda, H
中科院分区：
文献类型：
--
作者：
Kojima, M;Ayabe, K;Ueda, H
虽然构建融合蛋白或标记蛋白是获得双功能蛋白如具有特异性结合活性的酶的有用方法，但蛋白的适合融合的区域限于多肽的N-或C-末端，这常常妨碍其实用性。在这里，我们提出循环置换作为一种方法，用于将其他蛋白质拴在两个末端以外的位点。由于环状排列对实际重要蛋白质活性的影响尚待确定，因此大肠杆菌碱性磷酸酶在活性位点附近的环处进行环状排列，其新末端通过柔性接头连接。虽然在原始残基407和408处具有末端的置换体没有活性，但在残基90和94处具有末端的置换体显示出显著的活性。此外，在位置91和93以及外部肽表位处添加随机化残基产生了几种突变体，其比活性与具有类似外部肽的野生型酶相当。此外，突变体保留了与抗表位抗体的特异性结合活性，显示了它们在竞争性免疫测定中的潜在用途。
While the construction of fusion or tagged proteins is a useful method to obtain bifunctional proteins such as enzymes with specific binding activities, the region of the protein amenable to the fusion is limited to either the N- or C-terminus of the polypeptide, which often hampers its utility. Here we propose circular permutation as a method for tethering other protein(s) at a site(s) other than the two termini. As the effect of circular permutation on the activity of practically important proteins remains to be established, Escherichia coli alkaline phosphatase was subjected to circular permutation with its novel termini at the loops near the active site, and the original termini were linked by a flexible linker. While a permutant with the termini at original residues 407 and 408 was not active, a permutant with termini at residues 90 and 94 showed significant activity. Also, the addition of a randomized residue at positions 91 and 93 as well as outer peptide epitopes yielded several mutants with specific activity comparable to the wild-type enzyme with similar outer peptides. In addition, the mutants retained specific binding activity to anti-epitope antibodies, showing their potential utility in competitive immunoassay.