| Budget Amount *help |
¥14,900,000 (Direct Cost: ¥14,900,000)
Fiscal Year 2004: ¥7,300,000 (Direct Cost: ¥7,300,000)
Fiscal Year 2003: ¥7,600,000 (Direct Cost: ¥7,600,000)
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| Research Abstract |
Peptides, which are small biocompatible molecules with simple structure as compared to proteins, have shown various applications functioning as an antimicrobial molecule, inhibitory drug, targeting molecule, growth factor, and nano-material for biocompatible matrixes. Peptide arrays, developed as the SPOT method by R.Frank, have shown great efficiency in high-throughput screening of peptides for biomedical and pharmaceutical researches. In the SPOT method, activated amino acid monomers are spotted on support, such as cellulose membrane, to form a spatially addressed peptide library with the objective peptide length and order. In this study, we demonstrate the efficiency of our screening strategy in which a peptide array was used for direct peptide-cell interaction. In addition, the data obtained were also analyzed by computational science to establish the new method for functional peptide design, "designable proteomics". Consequently, the following results were obtained.
1)Screening of
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newry functional peptides
Peptide arrays covering the Ang II receptor type 1 sequence were prepared, and the peptide (VVIVIY) within the first transmembrane region exhibited the highest affinity to Ang II. The synthesized soluble VVIVIY peptide bad an 84% inhibitory effect on Ang II-induced aorta contration. In addition, we investigated the efficiency of the use of peptide array for direct cell assay, without cleavage of peptides, to design novel tumor growth-inhibitory peptides. From human Fas antigen ligand sequence, we found a novel 5-mer peptide domain (CNNLP) that strongly inhibits the growth of cancer cells by interaction with cell surface.
2)Analysis of peptide chip data by computational science to establish "designable proteomics"
2-1)HMM model construction for peptide screening
Hidden Markov model was constructed for screening of peptide combined with MHC class II molecule. The prediction acuracy of the model constructed was more than 90%.
2-2)Design of cell death inducing peptide
The effect of substitution of one peptide in the sequence of CNNLP was investigated by peptide chip cell assay system. From computational analysis of the data, the following rule was obtained to design the functional peptide ; 3 amino acid residues are important for the function and N-terminal amino acid should be small. Especially, threonin or valine is effective.
2-3)Exploring of lipases with inverted enantioselectivity for a substrate
We explored lipases with inverted enantioselectivity from the wild-type bacterial lipase, which is originally selective for (S)-configuration of the substrate. Computational tool constructed by us predicted a rule indicating that "size at position L167," among 4 positions(L17,F119,L167,and L266) in the substrate binding core region, is the most influential factor. Based on the guidelines obtained, we found two engineered novel variants, namely FIGV(L17F,F119I,L167G,and L266V) and FFGI(L17F,L167G,and L266I).
3)Exploring of cancer vaccine peptide
From the sequence of HSP70,the exploring of candidates of cancer vaccine peptide was investigated. HSP70 was one of the cancer specific proteins and a part of protein sequence is possible to become cancer vaccine peptide. From the assay of peptide chip spotting HSP70 sequence, more than 10 candidate peptide was obtained. Less
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