PROTEIN ENGINEERING OF ANTIBODIES FOR CREATING NOVEL MOLECULAR RECOGNITION PROPERTIES INTO AN ANTIBODY

抗体的蛋白质工程，将新颖的分子识别特性转化为抗体

基本信息

批准号：
06454668
负责人：
KUMAGAI Izumi
金额：
$ 4.54万
依托单位：
TOHOKU UNIVERSITY (1995-1996)The University of Tokyo (1994)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06454668/
关键词：
FILAMENTOUS PHAGE ANTIBODY MORECULAR RECOGNITION LYSOZYME PROTEIN ENGINEERING タンパク質

项目摘要

The display of foreign proteins on the surface of filamentous bacteriophages by fusing them to a minor coat protein of the phage (pIII), and subsequent selection of the phage with target proteins (by panning) has provided a powerful means of making novel functions into a protein. The results of this research can be summarized as follows : (1) I have focused on the interaction between hen egg-white lysozyme (HEL) and its monoclonal antibody, HyHEL 10. The variable region fragment (Fv fragment) of HyHEL 10 was prepared using bacterial expression system. A precise analysis that combined site-directed mutagenesis and titration calorimetry was performed, and several novel knowledge concerning the mechanism of antigen-antibody interaction could be clarified (Tsumoto et al., 1995,1996). (2) The variable domains of HyHEL 10Fv fragments were linked covalently with a flexible linker. A marked difference in the level of expression in E.coli was observed between VH-linker-VL (scFvHL) and VL-linker … More -VH (scFvLH). This HyHEL 10 scFvLh showed reduced binding activity toward its antigen, HEL,in comparison with Fv. Thermodynamic study showed that this reduced activity was due to entropic loss upon binding to its antigen, although this interaction between scFvLH and its antigen was enthalpically favorable. (3) For convenient analysis of antigen-antibody interactions, a novel phage display system has been developed (Maenaka et al., 1996), and using this system, the HyHEL10 single-chain varible region (scFv) fragment and the variable region of the heavy chain (VH) could be stably displayd on a filamentous phage. (4) HyHEL10 weakly recognizes human lysozyme (hL). To enhance the affinity of the Fv fragment toward hL,some of the residues in the heavy chain complementarity determining region 2 (HCDR2) of the HyHEL10 heavy chain varible region (VH) were randomized and the mutant library was subjected to selection using phage display technology. Several clones which bound to hL significantly were selected from the HCDR2 library, and these Fv fragments had increased affinity toward hL (Tsumoto et al., submitted). Less

通过将外源蛋白与噬菌体的次要外壳蛋白 (pIII) 融合，在丝状噬菌体表面展示外源蛋白，并随后用目标蛋白选择噬菌体（通过淘选），这提供了一种将新功能转化为本研究的结果可概括如下：（1）我重点研究了鸡蛋清溶菌酶（HEL）与其单克隆抗体HyHEL 10之间的相互作用。利用细菌表达系统制备了HyHEL 10的可变区片段（Fv片段），并进行了结合定点诱变和滴定量热法的精确分析，并且可以阐明有关抗原抗体相互作用机制的一些新知识（Tsumoto）。 (2)HyHEL 10Fv片段的可变结构域与柔性接头共价连接，表达水平存在显着差异。在大肠杆菌中观察到，与 Fv 相比，HyHEL 10 scFvLh 对其抗原 HEL 的结合活性降低。这种活性降低是由于熵，尽管在与其抗原结合时损失，但 scFvLH 与其抗原之间的这种相互作用在熵方面是有利的 (3) 为了方便分析。为了解决抗原抗体相互作用的问题，开发了一种新型噬菌体展示系统（Maenaka et al., 1996），利用该系统，HyHEL10单链可变区（scFv）片段和重链可变区（VH）可以(4)HyHEL10弱识别人溶菌酶(hL)以增强Fv片段对的亲和力。 hL，HyHEL10重链可变区（VH）的重链互补决定区2（HCDR2）中的一些残基被随机化，并使用噬菌体展示技术对突变体文库进行选择，获得与hL显着结合的几个克隆。从 HCDR2 文库中选择，这些 Fv 片段对 hL 的亲和力增加（Tsumoto 等人提交，Less）。