人源SIRT4蛋白在毕赤酵母中的表达及纯化
Expression and purification of human SIRT4 protein in Pichia pastoris

目的:获得表达量较高且有活性的人源SIRT4蛋白。方法:将SIRT4基因克隆到pPICZA载体上,得到重组pPICZA-SIRT4表达质粒。重组质粒线性化后转入到巴斯德毕赤酵母X33感受态细胞中,筛选出阳性菌株。将阳性重组菌株发酵培养,并对诱导剂甲醇浓度进行优化,表达蛋白用镍柱HisTrapTMHP进行纯化,并对表达的蛋白进行活性考察。结果:SDS-PAGE显示表达的SIRT4目的蛋白大小约33 000,与预期蛋白相对分子质量大小相符,进一步利用His标签抗体通过Western blot确认了目标蛋白,并且初步验证了SIRT4的去生物素酰化和去硫辛酰化的活性。结论:成功用巴斯德毕赤酵母表达了有活性的SIRT4蛋白,并且获得酵母表达的最佳诱导条件。
Aim: To obtain high-expression and active human SIRT4 protein.Methods: The SIRT4 gene was cloned into pPICZA vector to obtain the recombinant plasmid pPICZA-SIRT4. The recombinant plasmids were transformed into Pichia pastoris X33 competent cells after being linearized, and the positive strains were screened out. The positive recombinant strain was fermented and cultured, and the methanol concentration of the inducer was optimized. The expressed protein was purified with Ni column HisTrapTMHP and the activity of the expressed protein was investigated.Results: SDS-PAGE showed that the expressed protein SIRT4 was about 33 000, which was consistent with the predicted protein molecular weight. The target protein was further confirmed by Western blot using His-tag antibody. Finally, the debiotinylation and desulfonylation activity of SIRT4 was inspected.Conclusion: The active SIRT4 protein has been successfully expressed with Pichia pastoris and the optimal conditions for the yeast expression has been obtained