共表达蛋白质折叠辅助因子对毕赤酵母分泌表达IFNβ-HSA融合蛋白质的影响

Effect of Co-Expression of Protein Folding Factor on Expression of Fusion Protein IFNβ-HSA in Pichia pastoris

摘要:

探索共表达蛋白质折叠辅助因子Ero1、PDI和BiP对毕赤酵母GS115分泌表达融合蛋白质IFNβ-HSA的影响。将构建的蛋白质折叠辅助因子表达载体pGAP-Ero1、pGAP-PDI、pGAP-BiP和空载对照线性化后, 电击转化重组毕赤酵母GS115/IFNβ-HSA细胞, 用含有400 μg/mL zeocin的YPD平板筛选阳性转化子, 并采用PCR和Western blot法进一步鉴定。阳性转化子进行摇瓶发酵后, 采用SDS-PAGE及Western blot法分析共表达Ero1、PDI和BiP对IFNβ-HSA表达水平的影响。结果表明, Ero1、PDI和BiP成功地在胞内过量表达, 且不影响宿主细胞的正常生长; 共表达Ero1和PDI分别使IFNβ-HSA的表达量提高了80%和90%, 而共表达BiP则对IFNβ-HSA的表达水平无明显影响。

Abstract:

To explore the expression level of co-expression of folding factors Ero1, PDI and BiP on production of IFNβ-HSA by Pichia pastoris GS115. The pGAP-Ero1, pGAP-PDI, pGAP-BiP expression plasmids and the empty plasmid pGAPZ B(control) were linearized and integrated into the genome of P.pastoris GS115-IFNβ-HSA by electroporation. The recombinant yeasts were screened with 400 μg/mL zeocin. Positive recombinants were identified by genomic PCR and Western blot. The transformants were fermented in flask and the expression products were confirmed by SDS-PAGE and Western blot. The expression levels of IFNβ-HSA co-expressed with Ero1, PDI and BiP were analysed. The results indicated that Ero1, PDI and BiP were successfully expressed in recombinants with no effect on growth. Compared with Bip, co-expressed with Ero1 and PDI significantly improved the expression level of IFNβ-HSA, the expression level was 80%(Ero1) and 90%(PDI) higher than control.