CAJ | 학술논문

目的：通过对毕赤酵母中试发酵工艺的改进，建立一种简便可行的重组低出血抗凝蛋白（EH）的中试发酵工艺，为EH蛋白的放大生产研究奠定基础。方法：首先通过摇瓶培养绘测毕赤酵母工程菌的生长曲线，然后根据生长曲线，将对数生长期的菌种经过两级摇瓶培养放大后，直接接种到500 L的发酵罐中放大培养，通过发酵液的D600nm值、溶氧值（DO2）及菌体湿重动态监测细菌的生长状态，并用流加甲醇的方法诱导表达目的蛋白；表达上清经超滤、两步离子交换层析纯化获得目的蛋白；用非还原型SDS-PAGE和HPLC检测目的蛋白的纯度；用SDS-PAGE和质谱方法分析目的蛋白的相对分子质量；用Western印迹验证目的蛋白；用凝块法检测目的蛋白的抗凝活性。结果：发酵结束时，上清中蛋白含量达1.41 g/L，经后期分离纯化，得到约21 g EH蛋白，SDS-PAGE分析可见EH蛋白在还原状态下表观相对分子质量约为13.2×103±0.2×103，质谱分析相对分子质量约为7.3×103±0.73×103；Western印迹表明检测条带为目的蛋白，能被抗水蛭素抗体特异性结合；非还原型SDS-PAGE和HPLC测得EH蛋白的纯度均高于95%；凝块法检测EH蛋白的抗凝比活性为512~1024 ATU/mg。结论：建立了一条简便可行的EH蛋白的中试放大发酵生产工艺。
목적：통과대필적효모중시발효공예적개진，건립일충간편가행적중조저출혈항응단백（EH）적중시발효공예，위EH단백적방대생산연구전정기출。방법：수선통과요병배양회측필적효모공정균적생장곡선，연후근거생장곡선，장대수생장기적균충경과량급요병배양방대후，직접접충도500 L적발효관중방대배양，통과발효액적D600nm치、용양치（DO2）급균체습중동태감측세균적생장상태，병용류가갑순적방법유도표체목적단백；표체상청경초려、량보리자교환층석순화획득목적단백；용비환원형SDS-PAGE화HPLC검측목적단백적순도；용SDS-PAGE화질보방법분석목적단백적상대분자질량；용Western인적험증목적단백；용응괴법검측목적단백적항응활성。결과：발효결속시，상청중단백함량체1.41 g/L，경후기분리순화，득도약21 g EH단백，SDS-PAGE분석가견EH단백재환원상태하표관상대분자질량약위13.2×103±0.2×103，질보분석상대분자질량약위7.3×103±0.73×103；Western인적표명검측조대위목적단백，능피항수질소항체특이성결합；비환원형SDS-PAGE화HPLC측득EH단백적순도균고우95%；응괴법검측EH단백적항응비활성위512~1024 ATU/mg。결론：건립료일조간편가행적EH단백적중시방대발효생산공예。
Objective: To establish a practical method of pilot-scale yeast fermentation for the production of a low bleeding anticoagulant protein recombinant-EPR-hirudin(EH). Methods:The growth curve of recombinant Pi?chia pastoris secreting EH in shake flask cultivation was determined. Then, according to the determined growth curve, strains were amplified by two shake flask cultures and were directly inoculated into fermentation broth of 500 L. The fermentation was dynamically monitored by the dissolved oxygen and absorbance in 600 nm of the broth, and the wet weight of the strains as well. The target protein expression was induced with methanol feeding. After fermentation, the target proteins were purified through ultrafiltration and a two-step ion-exchange chromatogra?phy. The purity of target proteins was analyzed by SDS-PAGE and HPLC, and the molecular weight was exam?ined by reducing SDS-PAGE and mass spectrometry. Furthermore, EH was identified by Western blotting, and its anticoagulant activity was analyzed by a clot method. Results: After direct inoculation from flask cultivation, the re?combinant engineering yeasts grew well in a fermenter of 500 L tank volume. The total protein content of fermenta?tion supernatants was 1.41 g/L at the end of the fermentation. After separation and purification, 21 g of EH pro?tein was obtained. The results of the non-reducing SDS-PAGE and HPLC analysis showed that the purity of EH was more than 95%. The relative molecular weight of EH was about 7.3±0.73 kD determined by mass spectrome? try analysis, however, displayed about 13.2±0.2 kD under the SDS-PAGE analysis. The result of Western blotting indicated that the EH protein can be recognized by the commercial mouse anti-hirudin antibody. Moreover, the an?ti-thrombin activity of EH protein was about 512~1024 ATU/mg after incubation with bovine coagulation factor Xa. Conclusion: We have developed a simple and convenient method of pilot-scale yeast fermentation for the manufacture of EH.