Screening Of Affinity Peptide Ligands And Their Affinity Chromatography Of Proteins

This paper described the work of screening phage display peptide library to obtain high affinity ligand for some desired proteins and purifying the proteins by affinity column that modified with peptide ligands.An efficient procedure for the selection of high affinity clones from a heptapeptide phage display library was developed. Lysozyme was used as a model protein to demonstrate the selection strategy. The elution procedure was improved by alternating eluting the bound phages with glycine-HCl buffer (pH2.2) and high-concentration target protein solution. The modified method was compared with others, and the results confirmed that the modified procedure could yield high affinity phages that might be lost by other screening methods.Then, other proteins were used as target proteins to screening the peptide library with alternating elution strategy, and the phage showing high affinity to insulin or RNase A was obtained. Some phages showing low, medium, high specificity to each target protein, lysozyme, insulin and RNase A, had been selected respectively for sequencing. The results showed that the displaying peptides of the clones showing high specificity to each protein have the same amino acid sequence, that is HWWWPAS, and the phenomenon was analyzed. In additional, the affinity mechanism between the peptides with different specificity and those target proteins was analyzed.The peptide was synthesized and immobilized on EAH Sepharose 4B to prepare an affinity column. The chromatography experiments with lysozyme, human insulin, α-amylase and ribonuclease A as target proteins were carried out to examine their retention behaviors. It was found that the column exhibited the highest affinity only for insulin, and lysozyme can be retained partly, whereas the other two proteins were hardly retained. So, the binding mechanism of insulin to the peptide ligand was extensively studied using elution buffers of different pH values and with the addition of urea and ethylene glycol. As a result, electrostatic interactions and hydrogen bond were identified to contribute to the affinity interaction, while hydrophobic interaction contributed little. Moreover, the importance of electrostatic interactions and hydrogen bond to the affinity changed with pH value.The effect of rate of mobile phase on the retention of insulin was researched and the dynamic adsorption isotherm was got by analyzing the breakthrough data with Langmuir equation. Moreover, insulin was purified from the model mixture protein solution containing RNase A, α-amylase and insulin and from the crude extract solution of pig pancreas by the peptide affinity column, and the purity of the insulin was increased from 37% to 90% with 87.5% recovery.Because lysozyme also could be retained on the column, the experiment of purification of lysozyme from egg white with the column was carried out. After purified, the lysozyme activity factor was 7.3 higher than before with 87.3% recovery of the enzyme activity.