通过单独表达蛋白质分子伴侣二硫键异构酶(PDI)和共表达PDI和内质网氧化还原酶(Ero1),提高重组葡萄糖氧化酶在毕赤酵母中的分泌表达。将构建的蛋白质分子伴侣表达载体p PICZ/PDI和p PICZ/Ero1-PDI线性化后,电击转化重组毕赤酵母X33/p MD-GOD细胞,用含有250μg/m L G418和50μg/m L Zeocin的YPD双抗平板筛选阳性转化子,阳性转化子进行试管发酵和10 L发酵培养后,分析共表达PDI和Ero1-PDI对GOD表达水平的影响。结果显示,共表达PDI及Ero1-PDI分别使葡萄糖氧化酶在10 L发酵罐中30℃培养,酶活分别达到476 U/m L和736 U/m L,相比原始菌株在相同条件下分别提高了29.7%和100%。整合分子伴侣PDI和Ero1促进蛋白正确折叠明显地提高葡萄糖氧化酶蛋白表达。
【英文摘要】
The expression of recombinant glucose oxidase(GOD)in Pichia pastoris was enhanced by the sole expression of proteinchaperones protein disulfide isomerase(PDI)as well as the co-expression of endoplasmic reticulum oxidoreductase 1(Ero1)and PDI.The p PICZ/PDI and p PICZ/PDI-Ero1 expression plasmids were linearized and integrated into the genome of P. pastoris X33/p MD-GOD byelectroporation. The positive transformants were screened with double-resistance plate of 250 μg/m L G418 and 50 μg/m L Zeocin. The positi...
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