Semisynthetic Protein Probes for Protein Detection by Signal Amplification

• Main Authors: Lin, Hung sheung, 林弘盛
• Other Authors: Tan, Kui-Thong
• Format: Others
• Language: zh-TW
• Published: 2014
• Online Access: http://ndltd.ncl.edu.tw/handle/84539525416166578538

碩士 === 國立清華大學 === 化學系 === 102 === Protein plays an important role in our body. Enzyme is a kind of protein. It can regulate metabolism. Many disease is due to deregulatory of protein. Thus, detection of protein is an important issue. Nowadays, there are two famous detection methods. One is enzyme-linked immunosorbent assay (ELISA)which utilizes antibody to obtain specific binding for protein detection. The other is Western blot. It combines gel electrophoresis to separate protein and detects by antibody. This two methods are time consuming , high cost and require multi-steps operation. In this thesis, we described a new enzyme amplified method for protein detection based on semisynthetic protein sensor. Through molecular biology and organic synthesis, we constructed a recombinant protein SNAP-HCA and a synthetic compound. As a proof-of-principle, we will demonstrate the design of our semisynthetic protein sensor for the detection of avidin. The function of SNAP-tag protein is to facilitate specific labeling of the the synthetic compound to the recombinant protein, while the function of HCA is to regulate enzyme amplification during protein detection. The synthetic compound consists of three functional units and they are benzylguanine (BG) for specific labeling to the recombinant protein via SNAP-tag, biotin group for the binding and recognition with avidin, and a sulfonamide inhibitor for the regulation of HCA activity for signal amplification. In the absence of avidin, the protein sensor will exit in a closed conformation which will prevent the HCA enzyme to catalyze the hydrolysis of nonfluorescent FLDA to strongly fluorescent FL product. In the presence of avidin, the protein will bind to biotin and generate steric effects which should open up the protein sensor. Under this circumstance, the affinity of sulfonamide and HCA is weakened and the signal amplification can thus occur.