Kex2蛋白酶在毕赤酵母中的表达、纯化和性质研究

Expression,purification and properties of recombinant Kex2 in Pichia Pastoris

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。

Objective To study Kex2（ EC. 3. 4. 21. 61） expression,in Pichia Pastoris,purification and properties of the purified recombinant Kex2 proteinase. Methods The gene encoding Kex2 was cloned in p PIC9 K plasmid and transformed into GS115 by electroporation. After screened recombinant yeast strain with G418,induced yeast strain with methanol and analyzed with SDS-PAGE and activity detection,the recombinant Kex2 was successfully obtained. The secreted protein was purified with anion exchange chromatography（ Q-FF）,and some properties of the purified recombinant Kex2 was investigated. Results Molecular weight of Kex2 was about 67 k Da in SDS-PAGE,total activity recovery rate of Q-FF purification was 84%. The recombinant Kex2 was stable from p H 5. 0 to p H 6. 0,and optimal p H of recombinant Kex2 was p H 9. 0. 88. 2% activity was remained at 25 ℃,p H 5. 0for 12 h. Km was 0. 0167 mmol / L and Vmax was 333. 3 μmol / min with Boc-Gln-Arg-Arg-p NA as substrate. Conclusion The recombinant Kex2 is expressed successfully in Pichia yeast,and purified Kex2 is obtained after purification with anion exchange chromatography.