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

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.