| Budget Amount *help |
¥7,100,000 (Direct Cost: ¥7,100,000)
Fiscal Year 1996: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1995: ¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1994: ¥3,800,000 (Direct Cost: ¥3,800,000)
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| Research Abstract |
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
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-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
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