Construction and structural modeling of a single-chain Fv-asparaginase fusion protein resistant to proteolysis
Construction and structural modeling of a single-chain Fv-asparaginase fusion protein resistant to proteolysis
复制标题
抗蛋白水解的单链 Fv-天冬酰胺酶融合蛋白的构建和结构建模
DOI：
10.1002/1097-0290(20001120)70:4
复制
发表时间：
2000-11-20
影响因子：
3.8
通讯作者：
Meng, GZ
中科院分区：
文献类型：
--
作者：
Guo, L;Wang, JH;Meng, GZ
在这项研究中，我们构建了一个融合蛋白，该蛋白由来自大肠杆菌AS 1.357的l -天冬酰胺酶（ASNase）和保护性单链Fv （scFv）组成，该单链Fv是从我们之前研究的噬菌体展示scFv文库中选择的。融合蛋白的抗体片段通过连接肽（Gly(4)Ser）(6)与酶片段的n端融合。构建重组质粒pET-SLA，在大肠杆菌中高水平表达scFv-ASNase融合，表达产物形成包涵体。我们通过重折叠和纯化得到了可溶性融合蛋白。在相同的摩尔浓度下，可溶性融合蛋白的酶活性约为天然ASNase的82%，对底物l -天冬酰胺的K-m值与天然酶相似。重要的是，该融合蛋白比天然ASNase更稳定。此外：(1)经胰蛋白酶、a-凝乳胰蛋白酶和凝血酶在37℃下处理30 min后，scFv-ASNase分别保持了其原始活性的94.0%、88.8%和84.5%，而天然ASNase则失去活性；(2) ScFv-ASNase在血清中的体外半衰期（9 h）比天然酶（2 h）长得多。利用INSIGHT II软件包中的Homology和Discover模块进行建模，得到融合蛋白的三维结构。基于结构证据和生化特性，我们提出融合蛋白的scFv片段可能由于空间位阻和酶的静电表面变化而赋予ASNase片段对蛋白质水解的抗性。(C) 2000 John Wiley & Sons, Inc。
In this study, we construct a fusion protein composed of L-asparaginase (ASNase; from Escherichia coli AS 1.357) and a protective single-chain Fv (scFv), which was selected from a phage-display scFv library from our previous studies. The antibody moiety of the fusion protein was fused to the N-terminus of the enzyme moiety via a linker peptide, (Gly(4)Ser)(6). Recombinant plasmid pET-SLA was constructed to express scFv-ASNase fusion to high levels in E. coli and the expressed product was found to form inclusion bodies. We obtained a soluble fusion protein by refolding and purification. The soluble fusion protein exhibited about 82% of the enzymatic activity of the native ASNase at the same molar concentration, and had a K-m value similar to that of the native enzyme for the substrate L-asparagine. Importantly, the fusion protein was more stable than native ASNase. In addition: (1) following treatment with trypsin, a-chymotrypsin, and rennet, at 37 degreesC for 30 min, scFv-ASNase fusion retained 94.0%, 88.8%, and 84.5% of its original activity, respectively, whereas native ASNase became inactive; and (2) ScFv-ASNase fusion had a much longer in vitro half-life (9 h) in serum than the native enzyme (2 h). The three-dimensional structure of the fusion protein was obtained by modeling with the Homology and Discover modules of the INSIGHT II software package. On the basis of the structural evidence and biochemical properties, we propose that the scFv moiety of the fusion protein may confer ASNase moiety resistance to proteolysis as a result of both steric hindrance and a change in the electrostatic surface of the enzyme. (C) 2000 John Wiley & Sons, Inc.