Preparation Of Column Filling Of Multiple-ligand Affinity Chromatography

High-performance Affinity Chromatography(HPAC) is a method combined with classical affinity chromatography in an HPLC system. It has advantages of rapid analysis, high column efficiency and high recovery. Affinity chromatographic column based on porous particles of 8μm P(GMA-EDMA) is applicable for the analysis and micropreparative separation of proteins. In this paper, we prepared multiple-ligand affinity resin in which P(GMA-EDMA) microspheres were used as the support, and dye ligand and metal ligand were incorporated into these microbeads for the separation and purification of proteins. The main work follows:1. Porous particles, P(GMA-EDMA) micropheres with diameter of 7μm were prepared by a single-step swelling and polymerization method, using polystyrene micropheres prepared by dispersion polymation as seed particles and GMA,EDMA as monomers. The synthesized monodisperse P(GMA-EDMA) resin possesses uniform,porous characteristics and high mechanic intensity.2. Procion blue MX-R and Ni²⁺were coupled with P(GMA-EDMA) microspheres through different ways, thus two novel multiple-ligand affinity resins Ni²⁺-IDA-PB-P(GMA-EDMA),Ni²⁺-PB-P (GMA-EDMA) were prepared.3. The multipe-ligand immobilized P(GMA-EDMA) beads were slurry packed into stainless steel column.The protein retention machanism on multiple-ligand affinity chromatography was explored by comparing protein retention behavior and adsorption ability on multiple-ligand affinity chromatography with on mono-ligand affinity chromatography. Ni²⁺-IDA-PB-P(GMA-EDMA) column was selected as the best column for protein separation and purification.Ni²⁺-IDA-PB-P(GMA-EDMA)column(10×0.46cm I.D.) were investigated in selectivity, stabilization, protein loading capacity and recovery et. al., using lysozyme as standard protein. On this column,lysozyme was well eluted under the selected experimental conditions. The eluent flow rate was set at 0.5 ml/min with injection volume of 20μL and the mobile phase linear gradient was set in 15 min from 0 to 1 mol/L sodium chloride in 20 mmol/L phosphate buffer at pH 6.86. The column has low column backpressure, good stability, high protein mass recovery, and was factual used for the separation of lysozyme from egg white and purification of cytochrome C from procine heart.