Chen Yong, Li Yang, Liu Peng, Sun Qun, Liu Zhu
Department of Biology, Shantou University, Shantou, 515063, Guangdong, China,
Biotechnol Lett. 2014 Apr;36(4):711-8. doi: 10.1007/s10529-013-1411-3. Epub 2013 Dec 10.
A weakness of using immobilized metal affinity chromatography (IMAC) to purify recombinant proteins expressed in Pichia pastoris is the co-purification of native proteins that exhibit high affinities for Ni-IMAC. We have determined the elution profiles of P. pastoris proteins 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 from Tetrapisispora phaffii and Torulaspora delbrueckii, respectively. A new P. pastoris expression 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.
使用固定化金属亲和色谱法（IMAC）纯化在毕赤酵母中表达的重组蛋白存在一个缺点，即会共纯化对镍-IMAC具有高亲和力的天然蛋白。我们已经确定了毕赤酵母蛋白的洗脱曲线，并研究了用100 mM咪唑洗脱时共纯化的天然蛋白。鉴定出了四种主要污染物：线粒体乙醇脱氢酶同工酶III（mADH）、核苷酸切除修复内切酶，以及假定蛋白TPHA_0L01390和TDEL_0B02190，它们分别是源自法夫四孢酵母和德巴利酵母的同源蛋白。构建了一种新的毕赤酵母表达菌株，通过基因破坏消除了主要污染物mADH。蛋白质污染物的总量减少了55%，且不影响细胞生长。本研究证明了使用蛋白质组学方法促进生物工艺优化的可行性。
