Screening The Ligands Of Alpha-amylase From The Phage Heptapeptide Library

This work studied on screening the high affinity ligands of Alpha-amylase from phage heptapeptide library,synthesizing the highest affinity ligands using Fmoc solid-phase peptide synthesis method and purifying Alpha-amylase by affinity column that modified with synthesized peptide ligands.An efficient method for the selection of high affinity clone from phage peptide library was employed that developd in the laboratory. Alpha-amylase was used as a target molecule for screening its affinity ligands from heptapeptide phage display library.Based on the traditional method,the elution procedure was improved by alternating eluting with acid and target molecule.compared with the traditional screening method,the modified strategy yielded higher peptide recovery and the selected phage clones showed higher signals of enzyme-linked immunosorbent assay.After obtaining the ligands of Alpha-amylase,all kinds of different affinity phage clones was selected respectively to DNA sequencing and the amino acid sequences of the affinity clones were determined. The Pro and Glu as key amino acids binding to alpha-amylase were analyzed and discussed by the motif similarity between the affinity peptides.The highest affinity peptide ligand was synthesized by Fmoc solid-phase synthesis method.Due to the existence of Pro, DKP formation lead to the synthensis failure. Sothe heptapeptide was improved into hexapeptide for avoiding the DKP formation and not losing affinity,which was synthesized with the same method.At last I compared the yield and purifity of two product and identified the validity using LC/MS/MS.The synthesized peptide was immobilized on EAH Sepharose 4B by EDC method.The chromatography experiments was carried out to determine Alpha-amylase retention behaviors.The binding mechanism to peptide ligands was extensively studied by adjusting elution buffers of different pH values and ion intensity and appending the urea and ethylene glycol.The result was concluded that the affinity adsorption of amylase and ligand was contributed to the overall function of electrostatic interactions,hydrogen bond and hydrophobic interaction.