| Objective：To establish a practical method of pilot-scale yeast fermentation for theproduction of a low bleeding anticoagulant protein (EPR-Hirudin, EH). Methods：Thegrowth curve of recombinant pichia yeast secreting EH in shake flask cultivation wasdetermined. Then, according to the determined growth curve, strains were amplifiedby two shake flask cultures and were directly inoculated into fermentation broth of500L. The fermentation was dynamically monitored by the dissolved oxygen andabsorbance in600nm of the broth, and the wet weight of the strains as well. Thetarget protein expression was induced with methanol feeding. After fermentation, thetarget proteins were purified through ultrafiltration and a two-step ion-exchangechromatography. The purity of target proteins was analyzed by SDS-PAGE and HPLC,and the molecular weight was examined by reducing SDS-PAGE and massspectrometry. Furthermore, EH was identified by Western blotting, and itsanticoagulant activity was analyzed by a clot method. Results: After directinoculation from flask cultivation, the recombinant engineering yeasts grew well in afermenter of500L tank volume. The total protein content of fermentation supernatantswas1.41g/L at the end of the fermentation. After separation and purification,21g ofEH protein was obtained. The results of the non-reducing SDS-PAGE and HPLCanalysis showed that the purity of EH was more than95%. The relative molecularweight of EH was about7.3kD±0.73kD determined by mass spectrometry analysis,however, displayed about13.2kD±0.2kD under the SDS-PAGE analysis. The result ofWestern blotting indicated that the EH protein can be recognized by the commercialmouse anti-hirudin antibody. Moreover, the anti-thrombin activity of EH protein wasabout512～1024ATU/mg after incubation with bovine coagulation factor Xa.Conclusion: We have developed a simple and convenient method of pilot-scale yeastfermentation for the manufacture of EH. |
PDF Full Text Request