The cDNA of soluble human tumor necrosis factor receptor I (sTNFRI) was inserted into fusion-protein expression plasmid pIGF of A. niger to construct fusion expression vector pHBC containing a KEX2 like protein proces...The cDNA of soluble human tumor necrosis factor receptor I (sTNFRI) was inserted into fusion-protein expression plasmid pIGF of A. niger to construct fusion expression vector pHBC containing a KEX2 like protein processing site designed on the fusion position. Extracellular protease-deficient strain of A. niger 3.795-1-23 was transformed with pHBC. Positive clone was estimated by Southern hybridization. SDS-PAGE for protein produced by re-combinant strain showed the distinctive expression band. Western blotting indicated that the secreted protein had immunoactivity of sTNFRI.展开更多
文摘目的研究Kex2（EC.3.4.21.61）蛋白酶在毕赤酵母中的重组表达、纯化和部分性质。方法 Kex2基因整合进p PIC9K质粒中,通过电转构建重组毕赤酵母GS115-Kex2,G418筛选多拷贝子,甲醇诱导得到目的蛋白。通过强阴离子交换层析Q-FF对重组蛋白进行纯化,对纯化后的蛋白进行酶学性质研究。结果通过毕赤酵母表达可以得到具有活性的重组Kex2蛋白酶,以Boc-Gln-Arg-Arg-p NA（叔丁氧羰-谷氨酰胺-精氨酸-精氨酸-对硝基苯胺,Boc-QRR-p NA）为底物检测酶的活性,发酵外液活性达270U/L,SDS-PAGE检测目的蛋白的分子量为67k Da,经Q-FF纯化后,目的蛋白的活性回收率为84%。重组Kex2蛋白酶在p H 5.0~6.0之间稳定,p H 9.0时活性最高。在25℃,p H 5.0条件下放置12 h活力可保持88.2%。以Boc-QRR-p NA为底物,测得重组Kex2蛋白酶的Km值为0.0167 mmol/L,Vmax值为333.3μmol/min。结论利用毕赤酵母可成功表达分泌Kex2蛋白,经Q-FF纯化后可得到较纯的Kex2蛋白酶。
文摘The cDNA of soluble human tumor necrosis factor receptor I (sTNFRI) was inserted into fusion-protein expression plasmid pIGF of A. niger to construct fusion expression vector pHBC containing a KEX2 like protein processing site designed on the fusion position. Extracellular protease-deficient strain of A. niger 3.795-1-23 was transformed with pHBC. Positive clone was estimated by Southern hybridization. SDS-PAGE for protein produced by re-combinant strain showed the distinctive expression band. Western blotting indicated that the secreted protein had immunoactivity of sTNFRI.
