CAJ | 학술논문

TEV蛋白酶由于其高酶切活性、酶切位点的专一性和酶切条件的宽容性而在蛋白分离和蛋白质组学研究等领域有着广泛的应用.目前获得TEV蛋白酶的方法是将克隆在质粒上的TEV基因在大肠杆菌表达菌株BL21(DE3)中实现过表达.但本方法有一定缺点,如需使用抗生素,这就可能在后续的纯化中引入杂质,菌株群体中不均一性而降低产量等.本研究通过重组工程方法将受T7强启动子驱动的,与麦芽糖结合蛋白MBP融合的TEV蛋白酶基因整合至BL21(DE3)的基因组上进行过表达. MBP-TEV在细胞内自剪切获得分离的TEV. Ni-NTA亲和层析得到纯化的TEV,产量可达4.2 mg/L.所分离的TEV表达出良好的酶切活性.本菌株有着以之大规模获得TEV的潜力,所建立的通过重组工程将外源基因整合至BL21( DE3)基因组的进行表达的方法也可成为通用的平台技术.
TEV단백매유우기고매절활성、매절위점적전일성화매절조건적관용성이재단백분리화단백질조학연구등영역유착엄범적응용.목전획득TEV단백매적방법시장극륭재질립상적TEV기인재대장간균표체균주BL21(DE3)중실현과표체.단본방법유일정결점,여수사용항생소,저취가능재후속적순화중인입잡질,균주군체중불균일성이강저산량등.본연구통과중조공정방법장수T7강계동자구동적,여맥아당결합단백MBP융합적TEV단백매기인정합지BL21(DE3)적기인조상진행과표체. MBP-TEV재세포내자전절획득분리적TEV. Ni-NTA친화층석득도순화적TEV,산량가체4.2 mg/L.소분리적TEV표체출량호적매절활성.본균주유착이지대규모획득TEV적잠력,소건립적통과중조공정장외원기인정합지BL21( DE3)기인조적진행표체적방법야가성위통용적평태기술.
Due to its high proteinase cleavage activity,specificity and effective in a wide range of conditions,Tobacco etch virus protease ( TEV ) has many applications, ranging from protein isolation to proteomics study. Currently, TEV is produced by plasmid-based overexpression in Escherichia coli BL21(DE3). Yet,the method has inherent disadvantages, for example,it needs antibiotic to maintain the plasmid which may bring impurities during the protein purification;and non-homogeneity of the strain population which may reduce the yield. Herein,we report the recombineering mediated in-tegration of MBP fused TEV gene under the strong T7 promoter into E. coli BL21 ( DE3 ) chromosome. Intracellular digestion of chromosomal based overexpression of MBP-TEV released TEV which was subsequently isolated though Ni-NTA affinity purification, the yield of TEV was up to 4. 2 mg/L. Purified TEV shows fine protease activity. The engineered strain has the potential to be used for large scale TEV purification,the established recombineering method can be a platform for the genome engineering and heterologus gene expression in E. coli BL21(DE3).