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

外蛋白通过将噬菌体的次要涂层蛋白融合（PIII）以及随后选择靶蛋白（通过Panning）将噬菌体选择的噬菌体融合来显示出外蛋白在丝状细菌噬细胞表面的表面表现出来，这为使新功能提供了一种有力的含义。这项研究的结果可以总结如下：（1）我专注于使用细菌表达系统制备的Hyhel 10的可变区域片段（FV片段）之间的相互作用。进行了精确的分析，即进行了组合定点的诱变和滴定量热法，并且可以阐明有关抗原抗体相互作用机理的一些新知识（Tsumoto等，1995，1996）。（2）将HYHEL 10FV片段的可变域与灵活的接头共价链接。在VH-linker-Vl（SCFVHL）和VL-linker…More -VH（SCFVLH）之间观察到E.COLI中表达水平的明显差异。与FV相比，该Hyhel 10 SCFVLH与其抗原HEL的结合活性降低。热力学研究表明，尽管SCFVLH与其抗原之间的这种相互作用非常有利，但这种降低的活性是由于与其抗原结合后的熵损失所致。（3）为了方便地分析抗原抗体相互作用，已经开发了一种新型的噬菌体显示系统（Maenaka等，1996），使用该系统，Hyhel10单链可变区域（SCFV）片段和重链（VH）的可变区域（VH）可以稳定地显示在细丝噬菌体上。（4）Hyhel10弱认识到人类溶菌酶（HL）。为了增强FV片段对HL的亲和力，重链完整性中的某些残差确定Hyhel10重链可变区域（VH）的区域2（HCDR2）是随机的，并且使用噬菌体显示技术对突变库进行选择。从HCDR2文库中选择了几个与HL的克隆，并且这些FV片段对HL的亲和力增加（Tsumoto等人，提交）。较少的