豆豉溶栓酶基因在毕赤酵母中的表达及其产物的纯化

将含有和不含前肽的豆豉溶栓酶编码序列在毕赤酵母中分别进行表达研究,发现在含有前肽的豆豉溶栓酶基因转化的毕赤酵母工程菌用甲醇诱导时,其培养液中可检测到较强的溶栓酶活性;而在不含前肽的豆豉溶栓酶基因转化的毕赤酵母菌用甲醇诱导时,培养液中未检测到溶栓酶活性.对毕赤酵母表达和分泌的豆豉溶栓酶进行了分离纯化,并对其纯化产物进行溶栓酶活性、SDS-PAGE电泳、抑制剂作用及免疫印迹分析.结果表明,分泌到培养液中的豆豉溶栓酶已经过剪切加工,其前肽已被切除,重组与天然的豆豉溶栓酶具有相同的溶栓酶活性,其所测定的各理化指标均一致.本研究实现了豆豉溶栓酶在毕赤酵母菌中成功表达,并首次直接证明了前肽对豆豉溶栓酶在毕赤酵母中分泌表达是必须的.图3表1参20

EXPRESSION AND PURIFICATION OF SUBTILISIN DFE (DOUCHI FIBRINOLYTIC ENZYME) GENE IN PICHIA PASTORIS

The gene coding for a novel fibrinolytic enzyme, subtilisin DFE (douchi fibrinolytic enzyme) from Bacillus amyliquefaciens, was previously reported and its intact coding region was also identified. In this study, the recombinant plasmids expressing pro-subtilisin DFE, and subtilisin DFE were first constructed and transformed into Pichia pastoris. Strong fibrinolytic activity was detected only in the culture supernatant of the methanol induced pro-subtilisin DFE-transformants, but not in that of the other kinds of transformants. This result implied that the pro-peptide was important for the secretion of subtilisin DFE in P. pastoris, and after secretion, the pro-peptide was subsequently removed to produce active mature subtilisin DFE. The recombinant subtilisin DFE was purified to homogeneity from the culture supernatant of the induced P. pastoris transformant by three-step chromatography. The results from fibrinolytic activity, SDS-PAGE and Western-blotting analysis showed that the purified recombinant subtilisin DFE was the same as its native form. Fig 3, Tab 1, Ref 20