Study On The Coupling Process Of Sod's Purification-modification

Molcular modification technology is one of the hotspots in the second development of protein drugs, which can solve lots of problems in applying the drugs, such as unstability and short half-life. However, there are also some deficiencies of this technology, the process is complicated with many steps and long periods. In this paper, SOD was used as a template, and the procedures of purification and modification were coupled to obtain more stable modified SOD with higher activity. And moreover, the feasibility of purification-modification coupling process in separating proteins was discussed.First of all, SOD's coupling procedure of IEC purification - PEG modification was studied, using DEAE 52 as solid phase medium. The adsorption and modification condition was optimized by orthogonal design. Absorption temperature was 25℃, pH was 7.8, solution concentration was 1.0 mg/ml. The adsorption capacity reached 5.3366×10⁴U/mg. SOD modified by mPEG(50 fold), at temperature 25℃, in borate buffer( pH 9.2, 50 mmol/L). PEG-SOD washed by NaCl (0-0.4 M) grads. As a result, the specific activity of PEG-SOD is 9 638 U/mg, with purification rate of 1.87. It was modified at 35.3% of its free amino groups.Secongdly, SOD's coupling procedure of MCAC purification - PEG modification was studied, using Cu²⁺-sephadex G75 as solid phase medium. The adsorption and modification condition was optimized by orthogonal design. Absorption temperature was 25℃, pH was 8.0, solution concentration was 1.5 mg/ml and contained 0.5 M NaCl. The adsorption capacity reached 9.69×10⁴ U/g. SOD modified by mPEG(50 fold), at temperature 25℃, in borate buffer(pH 9.2, 100 mmol/L). Then PEG-SOD was eluted by Tris-HCl buffer (pH6.0, 0.5 M NH₄Cl). As a result, the specific activity of PEG-SOD is 9 067 U/mg, with purification rate of 1.755. It was modified at 40.9% of its free amino groups, retained 88.43% of enzymatic activity of native SOD.Finally, partial properties of modified SOD were studied. The results showed that the stability of PEG-SOD was enhanced significantly and we found it feasible to couple purification process with modification process in separating process.