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.
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Acknowledgments
This work was supported by grants from National Natural Science Foundation of China (Nos. 31100052 and 31370163), Guangdong Natural Science Foundation (Nos. S2012010009941 and S2013010014006), Science and Technology Program of Guangdong Province (No. 2011B020308015), University Talent Introduction Plan of Guangdong Province (2011, No. 430), International Science and Technology Cooperation Project of China (No. 2011DFR31220-2), Science and Technology Program of Shantou City (Nos. G201200063 and D201200324), and Scientific Research Foundation for Returned Scholars, Ministry of Education of China (2012, No. 940). We are appreciated to Dr. Chiju Wei for revising this manuscript.
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Chen, Y., Li, Y., Liu, P. et al. Optimized expression in Pichia pastoris eliminates common protein contaminants from subsequent His-tag purification. Biotechnol Lett 36, 711–718 (2014). https://doi.org/10.1007/s10529-013-1411-3
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DOI: https://doi.org/10.1007/s10529-013-1411-3