Purification Of Insulin By Affinity Precipitation

Insulin is one kind of important medical protein hormone,only a few experimens have been done for its purification,so it’s necessary to find more effective purification methods.In this paper,it is the first time to study affinity precipitation method for purification of Insulin.One kind of new polymer is synthesized for affinity precipitation.It is random synthesized by adjusting the proportion of four kinds of monomers:methacrylic acid,methyl methacrylate,dimethylaminoethyl methacrylate,and N-hydroxymethyl acrylamide based on others’s experiments in our laboratory,azodiisobutyronitrile as initiator.The isoelectric point of the polymer is 3.51 by Zeta potential,the recovery of polymer is 96.1%by weight method,the molecular weight is126.3KDa by viscosity method,and the weight average molecular weight is 4.904 X 105Da by Gel permeation chromatography method.As ligand,zinc ion is connected to polymer while epichlorohydrin as activator and iminodiacetic acid as chelator.Ligand could reach the highest concentration 92.7μmol/g polymer when the activator is 0.9ml/g polymer.During the procedure of affinity precipitation,the optimal adsorption conditions are reacted for 150min,0.8MNa2SO4,pH7.5 of PBS,the adsorption recovery is higher with the rising of ligand concentration,and the maximum adsorption capacity is 153.86mg/g copolymer by Langmuir isotherm;the elution recovery could reach 96.2%by 0.8mmol/ml imidazole,0.5mmol/ml CH3COOH and 0.9mmol/ml CH5N3·HCl.The eluted insulin activity is measured by HPLC，and the activity recovery could reach 95.2%.Copolymer could be recycled for 5 times with adsorption 100mg/g and elution recovery more than 95%every time.These results have showed that affinity precipitation could be applied to the purification of insulin.