Optimized expression in Pichia pastoris eliminates common protein contaminants from subsequent His-tag purification
Optimized expression in Pichia pastoris eliminates common protein contaminants from subsequent His-tag purification
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DOI：
10.1007/s10529-013-1411-3
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发表时间：
2014-04
影响因子：
2.7
通讯作者：
Yong Chen;Yang Li;Peng Liu;Qun Sun;Zhu Liu
中科院分区：
文献类型：
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作者：
Yong Chen;Yang Li;Peng Liu;Qun Sun;Zhu Liu
利用固定化金属亲和层析（immobilized metal affinity chromatography，IMAC）纯化毕赤酵母表达的重组蛋白的一个缺点是对Ni-IMAC表现出高亲和性的天然蛋白的共纯化。测定了P的洗脱曲线。pastorisproteins，并检查了天然蛋白质，共纯化时，用100 mM咪唑洗脱。鉴定出4种主要的污染物：线粒体乙醇脱氢酶同工酶III（mADH）、核苷酸切除修复内切酶和假设的蛋白TPHA_0L01390和TDEL_0B02190，它们分别是来自Tetrapisispora phaffii和Torulaspora delbrueckii的同源蛋白。新来的。pastorisexpression strain被改造为通过基因破坏消除主要污染物mADH。蛋白质污染物的总量减少了55%而不影响细胞生长。本研究证明了使用蛋白质组学方法来促进生物过程优化的可行性。
A weakness of using immobilized metal affinity chromatography (IMAC) to purify recombinant proteins expressed inPichia pastorisis the co-purification of native proteins that exhibit high affinities for Ni-IMAC. We have determined the elution profiles ofP. pastorisproteins and have examined the native proteins that co-purify when eluting with 100 mM imidazole. Four major contaminants were identified: mitochondrial alcohol dehydrogenase isozyme III (mADH), nucleotide excision repair endonuclease, and the hypothetical proteins TPHA_0L01390 and TDEL_0B02190 which are homologous proteins derived fromTetrapisispora phaffiiandTorulaspora delbrueckii, respectively. A newP. pastorisexpression strain was engineered that eliminated the predominant contaminant, mADH, by gene disruption. The total amount of protein contaminants was reduced by 55 % without effecting cell growth. The present study demonstrates the feasibility of using a proteomic approach to facilitate bioprocess optimization.