title: Optimized expression in Pichia pastoris eliminates common protein contaminants from subsequent His-tag purification.
abstract: 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.
authors: Department of Biology, Shantou University, Shantou, 515063, Guangdong, China, 11ychen@stu.edu.cn.
authors: Yong
authors: Y
authors: Chen
authors: Yang
authors: Y
authors: Li
authors: Peng
authors: P
authors: Liu
authors: Qun
authors: Q
authors: Sun
authors: Zhu
authors: Z
authors: Liu
mesh_terms: D002846:Chromatography, Affinity; D017353:Gene Deletion; D015870:Gene Expression; D005822:Genetic Vectors; D060847:Metabolic Engineering; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D011994:Recombinant Proteins