| Erythropoietin is a glycoprotein hormone used to treat kidneys failure. All of engineered erythropoietin used in clinical produced by CHO cell culture. In order to decrease EPO production cost, Shanghai transgenic research center cloned hEPO gene and expressed rhEPO in high expression level in the mammary of transgenic goats which produced by nuclear transfer.In this paper, we studied the purification of rhEPO from the milk of transgenic goat and characterized the purified rhEPO.Firstly, after remove the lipids by centrifuge, different steps of pre-treatment of the milk were tested. At pH4.6, acid precipitated can move nearly almost caseins and have the rhEPO 97.8% recovery.Secondly, several chromatography methods such as the Ion-exchange Chromatography (IEC), Hydrophobic Interation Chromatography (HIC), reverse-phase chromotagraphy and Gel Filtration Chromatography (GF) were studied. During the experiments of purification of rhEPO with SP, the optimal pH of buffer and concentration of the salt were established, and the results showed the recovery of activity was 53.8% and 90% impurities were eliminated. The purification condition of HIC was optimized, 98% impurities were eliminated and the recovery of rhEPO was 28.7%. During the experiments of purification of rhEPO with GF, Superdex G75 was selected, the recovery activity was 66.6%, the specific activity increased 0.79 times. The recovery of total rhEPO was 10.1%.Finally, based on the above researches we decided the optimal procedure of purification rhEPO. The characters of purified rhEPO have been studied. The product gave a single broad band on SDS-PAGE and had an in vitro specific acitivity of 240,000 lU/mg. The purity of rhEPO was at least 98%. The pl of rhEPO is 6-8 identified by IEF. The make up amino acids of N-terminal of rhEPO were determined and consistent with the natural EPO. Allof the datas indicated the purification procedure was effective and high purity products could get from this technics. |
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