Combinatorial approach to investigate enzyme specificities of protease

• Main Authors: Hsiao Fei-ya, 蕭斐雅
• Other Authors: 張文章
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/33552878919873655977

碩士 === 國立臺灣大學 === 生化科學研究所 === 88 === The solid phase peptide synthesis method was proposed by Merrfield in 1963. The technology was not only used to make peptide synthesis faster and more simply, but also applied to many other fields. Most of all, solid phase synthesis was applied to synthesize combinatorial library recently. Combinatorial library is a powerful tool to find out active compounds from a mixture of thousands compounds. It is the efficient approach for finding the biologically active molecules. The main aim of the thesis is to establish a simple combinatorial peptide library for screening the substrate specificities of proteases. We constructed a library of tetrapeptide-ester in which all the compounds were just different in the P1 site. The protease will catalyze the hydrolysis of peptide-esters that have the suitable amino acid residue in the P1 site and release tetrapeptide from the peptide-ester library. By using high performance liquid chromatography as monitor, we could find out the peptide-ester with faster hydrolysis rate. According to the most suitable amino residue of the P1 site, the peptide-ester library in which were different in the P2 site was synthesized by the〝split-couple-mix〞method and repeat the protease-catalyzed hydrolysis reaction to screen the suitable amino acid residue of P2 site. Before the specificity studies of unknown protease, the accuracy of the library was tested by the proteases which their specificities were well known.The trypsin, chymotrypsin, endoproteinase Lys-C and endoproteinase Glu-C were chosen for the test, and the experimental results shown that the peptide-ester library we constructed was suitable for the enzyme specificity studies. After confirming the accuracy of the library, we used the library to study the protease TM-1 which the specificity was unknown and found that its specificity in P1 site prefers phenylalanine and tyrosine, both have phenyl group at their side chain. Based on the P1 site amino acid residue, we synthesized peptide libraries which changed the amino acid residue in the P2 site by〝split-couple-mix〞method and studied the P2 site of TM-1.We found that TM-1 prefers hydrophobic amino acid residues at P2 site. Overall, we show that the combinatorial library approach is a simple method to investigate specificity.