Research On Purification Of Recombinant Human-like Collagen Using Ion Exchange And Its Kinetics

Purification of recombinant human-like collagen bioprotein (HCB) was researched in this paper. Harvested recombinant E.coli cell was collected and disrupted by high pressure homogenizer, supernatant was collected by centrifuging. After precipitation, the crude product solution of HCB was purified by ion exchange chromatography, and a high purity could be reached.The capacities of adsorping impurityies of two cation exchange resins, CM-1 and CM52, were compared. The results showed that: the purification effect of CM-1 was better than that of CM52. The techniques of purification for HCB using CM-1 were confirmed. If the protein concentration was 4 mg·mL^-1, made by 20mmol·L^-1 phosphate buffer with 0.03 mol·L^-1 NaCl at pH6.5, the purity, which is 82.1%, and the recovery of HCB, which is 84.2%, could be reached after one hour' adsorption.Reaserches on the adsorption equilibrium of anionic ion exchanger, Fractogel EMD DEAE, showed that the adsorption of HCB onto Fractogel EMD DEAE obey Langmuir model. The value of pH and the concentration of NaCl were two important factors which affected the parameters of Langmuir model greatly and the effect of pH value on charecteristics of ion exchanger was complicated. With the pH value increasing, the parameter, K_d, decreased and the binding capacity added. Due to the limitations of properties of ion exchanger and HCB, pH value could not be too high. If more NaCl concentration was added, the binding capacity decreased and the parameter, K_d, increased. The experimental results showed that the influences of temperature on binding capacity and K_d were not so obviously.The purification process of HCB by Fractogel EMD DEAE was optimized with the data of adsorption equilibrium. The purity of 96.8% and the HCB recovery of 80.4% could be achieved on the condition that the HCB concentration with Tris-HCl buffer at pH 8.0 was 4 mg·mL^-1, which was adsorbed through anionic ion exchanger at 3.0 mL·min^-1 rate and eluted by stepwise elution.The results of ion exchange kinetics of the adsorption of HCB on Fractogel EMD DEAE showed that pore diffusion model could describe dynamic adsorption behaviors of HCB onto Fractogel EMD DEAE. The higher the ionic strength was, the higher the pore diffusion coefficient D_p,p was. The higher the initial protein concentration was, the lower the value of D_p,p was.