特异腐质霉角质酶-OMP25融合蛋白在大肠杆菌中的高效表达
作者:
  • 晏婷婷

    晏婷婷

    江南大学食品科学与技术国家重点实验室, 江苏 无锡 214122;江南大学生物工程学院工业生物技术教育部重点实验室, 江苏 无锡 214122;江南大学教育部食品安全国际合作联合实验室, 江苏 无锡 214122
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  • 刘展志

    刘展志

    江南大学食品科学与技术国家重点实验室, 江苏 无锡 214122;江南大学生物工程学院工业生物技术教育部重点实验室, 江苏 无锡 214122;江南大学教育部食品安全国际合作联合实验室, 江苏 无锡 214122
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  • 李光耀

    李光耀

    江南大学食品科学与技术国家重点实验室, 江苏 无锡 214122;江南大学生物工程学院工业生物技术教育部重点实验室, 江苏 无锡 214122;江南大学教育部食品安全国际合作联合实验室, 江苏 无锡 214122
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  • 吴敬

    吴敬

    江南大学食品科学与技术国家重点实验室, 江苏 无锡 214122;江南大学生物工程学院工业生物技术教育部重点实验室, 江苏 无锡 214122;江南大学教育部食品安全国际合作联合实验室, 江苏 无锡 214122
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基金项目:

国家重点研发计划(2019YFA0706900);江苏省科技厅政策引导类计划(国际科技合作/港澳台科技合作)—“一带一路”创新合作项目(BZ2020010)


High-efficiency expression of Humicola insolens cutinase-OMP25 fusion protein in Escherichia coli
Author:
  • YAN Tingting

    YAN Tingting

    State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu, China;Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China;International Joint Laboratory on Food Safety, Ministry of Education, Jiangnan University, Wuxi 214122, Jiangsu, China
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  • LIU Zhanzhi

    LIU Zhanzhi

    State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu, China;Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China;International Joint Laboratory on Food Safety, Ministry of Education, Jiangnan University, Wuxi 214122, Jiangsu, China
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  • LI Guangyao

    LI Guangyao

    State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu, China;Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China;International Joint Laboratory on Food Safety, Ministry of Education, Jiangnan University, Wuxi 214122, Jiangsu, China
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  • WU Jing

    WU Jing

    State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu, China;Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China;International Joint Laboratory on Food Safety, Ministry of Education, Jiangnan University, Wuxi 214122, Jiangsu, China
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    摘要:

    【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,pNPB)、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。

    Abstract:

    [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 (pNPB) and polyethyl acrylate (PEA) and stability at 50℃ were determined. In addition, the changes in the cell membrane permeability and cell surface hydrophobicity of host bacteria were detected in HiC-OMP25 expression, 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 pNPB was degraded. However, the degradation effect of the former on PEA and its stability at 50℃ 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 pNPB 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.

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晏婷婷,刘展志,李光耀,吴敬. 特异腐质霉角质酶-OMP25融合蛋白在大肠杆菌中的高效表达[J]. 微生物学报, 2022, 62(12): 4918-4926

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  • 收稿日期:2022-03-29
  • 最后修改日期:2022-05-06
  • 在线发布日期: 2022-12-08
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