重组低出血抗凝蛋白在毕赤酵母中的中试发酵工艺研究及其纯化与鉴定  

Pilot-Scale Fermentation Study of a Low Bleeding Anticoagulant Protein in Pichia pastoris

作　　者：郝木强[1,2] 李彦英[3] 刘春杰[4,2] 王秀冬[2] 刘晶晶[4,2] 李艳琪[4,2] 刘洋[4,2] 吴祖泽[2] 靳继德[2] 

机构地区：[1]北京工业大学生命科学与生物工程学院,北京100024 [2]军事医学科学院放射与辐射医学研究所,北京100850 [3]军事医学科学院生物工程研究所,北京100071 [4]天津大学化工学院,天津300072

出　　处：《生物技术通讯》2013年第3期314-319,共6页Letters in Biotechnology

基　　金：国家重大新药创制科技重大专项(2012ZX09102301-008)

摘　　要：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500L的发酵罐中放大培养，通过发酵液的D_600nm值、溶氧值（D02）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1．41g／L，经后期分离纯化，得到约21gEH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13．2×10^3±0．2×10^3，质谱分析相对分子质量约为7.3×10^3±0．73×10^3；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512～1024ATU／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。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 Pichia 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 chromatography. The purity of target proteins was analyzed by SDS-PAGE and HPLC, and the molecular weight was examined 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 inoeulation from flask cultivation, the recombinant engineering yeasts grew well in a fermenter of 500 L tank volume. The total protein content of fermentation supernatants was 1.41 g/L at the end of the fermentation. After separation and purification, 21 g of EH protein 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 spectrometry 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 anti-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.

关 键 词：水蛭素 重组蛋白 毕赤酵母 中试放大 

分 类 号：Q78[生物学—分子生物学]