特异腐质霉角质酶-OMP25融合蛋白在大肠杆菌中的高效表达

High-efficiency expression of Humicola insolens cutinase-OMP25 fusion protein in Escherichia coli

【目的】通过探究特异腐质霉角质酶-OMP25融合蛋白(HiC-OMP25)在不同大肠杆菌(Escherichia coli)菌株中的表达情况、底物降解情况、热稳定性及宿主菌细胞膜通透性与细胞表面疏水性,揭示表达HiC-OMP25时不同宿主菌的差异性,并进一步提高HiC-OMP25在大肠杆菌中的表达量。【方法】分别在E.coli BL21(DE3)及E.coli C43(DE3)中表达HiC-OMP25,并测定其对对硝基苯丁酸酯(4-nitrophenol butyrate,p NPB)、聚丙烯酸乙酯(polyethyl acrylate,PEA)的降解效果、50℃稳定性;测定表达HiC-OMP25时宿主菌的细胞膜通透性及细胞表面疏水性变化;共表达伴侣蛋白提高HiC-OMP25在E.coli C43(DE3)中的表达量。【结果】HiC-OMP25在E.coli BL21(DE3)与E.coli C43(DE3)中均成功表达并降解pNPB,但前者对PEA的降解效果及50℃稳定性均低于后者。同时,表达HiC-OMP25显著增强了E.coli BL21(DE3)的细胞膜通透性及细胞表面疏水性。HiC-OMP25与巯基氧化酶(Erv1p)、二硫键异构酶(DsbC)在E.coli C43(DE3)中共表达时,其表达量为原始菌株的2.14倍,且对pNPB及PEA均有良好的降解效果。【结论】异源表达时,HiC-OMP25在E.coli C43(DE3)中正确折叠,而在E.coli BL21(DE3)中未完全正确折叠;通过共表达伴侣蛋白提高了HiC-OMP25在E.coli C43(DE3)中的表达量,为以后HiC-OMP25的工业化生产及应用奠定了基础。

[Objective]To explore the expression,substrate degradation and thermal stability of Humicola insolens cutinase-OMP25 fusion protein(HiC-OMP25)in different Escherichia coli strains as well as the host cell membrane permeability and cell surface hydrophobicity,so as to reveal the differences in the expression of HiC-OMP25 by different host bacteria and further increase the expression of HiC-OMP25 in E.coli.[Methods]HiC-OMP25 was expressed in E.coli BL21(DE3)and E.coli C43(DE3),separately,and their degradation effect on 4-nitrophenol butyrate(p NPB)and polyethyl acrylate(PEA)and stability at 50°C were determined.In addition,the changes in the cell membrane permeability and cell surface hydrophobicity of host bacteria were detected in HiC-OMP25expression,and the expression of HiC-OMP25 in E.coli C43(DE3)was explored by co-expressing chaperone proteins.[Results]HiC-OMP25 was expressed in E.coli BL21(DE3)and E.coli C43(DE3)and p NPB was degraded.However,the degradation effect of the former on PEA and its stability at 50°C were both lower than those of the latter.Additionally,HiC-OMP25 significantly enhanced the cell membrane permeability and cell surface hydrophobicity of E.coli BL21(DE3).Co-expression of HIC-OMP25 with sulfhydryl oxidase(Erv1p)and disulfide isomerase(DsbC)in E.coli C43(DE3)finally increased the expression level of HIC-OMP25 by 2.14 times,well degraded p NPB and PEA.[Conclusion]When heterologously expressed,HiC-OMP25 folded correctly in E.coli C43(DE3),but not in E.coli BL21(DE3).Co-expression of chaperone proteins improved the expression of HiC-OMP25 in E.coli C43(DE3),which laid a foundation for the industrial production and application of HiC-OMP25 in the future.