| Summary: | 碩士 === 國立交通大學 === 應用化學系碩博士班 === 102 === The study is aimed to screen a single chain antibody fragment (scFv) from phage display library for cAMP recognition. The cAMP derivative (containing alkyl amine moiety) was conjugated on BSA and employed for scFv screening. After 6 rounds of panning, 3 clones (designated as 1D, 2E and 5H) with high titers were obtained. Since 2E exhibited higher titer than the other two clones, 2E clone, virtually composed of only the light chain variable region (VL), was mainly used in this study. The corresponding protein, named as cAMP-VL, was overexpressed and purified. The molecular weight was analyzed (14052.8 Da) by electrospray ionization mass spectrometry and confirmed to be consistent with the theoretical value (14054.3 Da). The binding features of cAMP-VL as free protein or on the surface of phage were further analyzed by ELISA or quartz crystal microbalance (QCM). The experiments were conducted using cAMP as probe for interacting with 2E phage and cAMP-VL. The detection limits of 2E phage and cAMP-VL are 200 phage particle/mL and 0.66 µg/mL, repectively. In the case of QCM analysis, cAMP derivative was immobilized on QCM chip, the dissociation constant (Kd) was estimated to be 74,900 phage particle/mL. In order to investigate the binding specificity of cAMP-VL, several nucleotides (adenosine,ATP,ADP and AMP) and nucleosides were used for competitive binding assessment. Results showed that cAMP-VL possesses high specificity to purine moiety. Further study on structure simulation attempted to sketch the binding domain of cAMP. P59, R61, and E81 were found to be the possible candidates for binding. P59R, R61K and E81Q mutants were constructed and purified for binding study. The outcome showed all three mutants moderately damaged the interaction with cAMP indicating these residues are somewhat important for cAMP binding. More studies will be proposed to improve the binding affinity of cAMP-VL toward cAMP based on the structure simulation or the complex of 3D protein structure.
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