Preparation Of Sepharose Affinity Adsorbent & Purification Of Trypsin

As an important purification method for protein, affinity adsorption has been widely used in biochemical and medical industries for its large production and high active recovery. The key of affinity adsorption is to prepare the affinity adsorbent with high specific, adsorption capability, good mechanical strength, heat and stress resistance. On one hand, the smaller the particle size is, the higher the activation degree got,and more affinity ligands can be coupled onto microspheres; on the other hand, high specific adsorption produces high activity recovery and purified fold. In traditional fixed bed adsorption, big pressure drop causes deformation of media, and the production scale is restricted. Suspension affinity adsorption, through suspension, adsorption, desorption, is investigated, to get protein with high purity and activity. This method is simple, repeatable,and provides basis data for further research on continuous affinity adsorption process.Currently, the particle size of Sepharose used in industry is 200-300μm. The wide specific surface area and adsorbance can be achieved through the small particle size and good monodispersity,besises, space arm should be bonded to reduced the steric hindrance. In this paper, specific mirco adsorbent was prepared, with research on the epichlorohydrin activation solven, the space arm; the specific ligand.The main, work included:Firstly,high specific adsorbent was prepared, using Sepharose CL-6B as micro carrier and through steps of pre-processing,activation solvent,space arm,specific ligand.Secondly, with trypsin as target protein, separation process was carried through affinity adsorption in adsorption buffer solution (pH7.9) oscillated for 1h at 30℃200rpm;and continuous elution for three times with KCl solution (0.01M acetic acid) as eluent. The adsorbent has an adsorption capacity of 33.3mg/g; yield of 83.70% and purified fold of 7.37. The suspension affinity adsorption conforms to Langmuir adsorption isotherm equation, can be expressed as besides, the nonspecific adsorption of BSA is 0.3mg/g,which is merely 0.75% for trypsin.Finally, Agarose-Dextran microspheres was prepared by inverse suspension method, based on which, complex affinity adsorbent was furtherly made.It is shown that the affinity adsorbent has good suspension affinity adsorption ability, with an adsorption capacity of 30.80mg/g; yield of 88.70% and purified fold of 6.88.