Identification of Interaction between Recombinant Horseshoe Crab Plasma Lectin and Lipopolysaccharide via MALDI-TOF MS

• Main Authors: Chou, Sheng-Hao, 周聖浩
• Other Authors: Wu, Tung-Kung
• Format: Others
• Language: en_US
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/52898897443640496872

碩士 === 國立交通大學 === 生物科技學系 === 104 === Recombinant horseshoe crab plasma lectin (rHPL) derived from Taiwanese Tachypleus tridentatus has been successfully expressed and purified in E. coli system, and has been proven to recognize pathogen associated molecular patterns (PAMPs) through rhamnose. Also, rHPL is proven to inhibit bacterial growth such as P. aeruginosa, which makes rHPL an appropriate prototype in the development of a novel AMP. The binding mechanisms between rHPL and PAMPs are not yet thoroughly comprehended. MALDI-TOF MS with highly sensitive and precise features is applied to verify the inhibitory binding between rHPL and four different lipopolysaccharides (LPS): Pseudomonas aeruginosa 10, Pseudomonas aeruginosa PAO1, Pseudomonas aeruginosa PA14 and Klebsiella pneumoniae separately. Our results indicated that all four LPSs were confirmed to interact with rHPL and could be identified in the MALDI-TOF MS spectrum. The finding of rHPL interacting with two infectious strains could facilitate the applicability of novel diagnostic and therapeutic strategies for microbial pathogens in future development. Also our novel approach to verifying molecular interactions using MALDI-TOF MS could be applied to other LPS-binding proteins. Besides, 3-dimensional structure of rHPL has not been solved. We use molecular docking technique to predict ligand binding sites of rHPL and, successfully verify that Y46 and F103 are key amino acids in ligand binding.