Inhibition of degradation and aggregation of recombinant human consensus interferon-alpha mutant expressed in Pichia pastoris with complex medium in bioreactor.
The methylotrophic yeast Pichia pastoris has been used for the expression of many proteins. However, limitations such as protein degradation and aggregation became obvious when secreting heterologous protein-recombinant human consensus interferon-alpha mutant. Here, we investigate the effect of induction temperature on the yield and stability of interferon mutant expressed by P. patoris with buffered complex medium. The best results in terms of interferon mutant bioactivity and specific bioactivity were obtained when the microorganism was induced at 15 degrees C, which were 2.91 x 10(8) +/- 0.3 x 10(8) and 2.26 x 10(8 )+/- 0.23 x 10(8) IU mg(-1), respectively. At the same time, the cells grew fast owing to high AOX1-specific activity, and interferon mutant expression level reached 1.23 g l(-1), which was almost 30 times higher than that in the flask. Also, the proteolytic degradation of interferon mutant was inhibited completely because of lower protease bioactivity probably due to a reduced cell death rate at lower temperatures as well as protection of yeast extract and peptone in complex medium. In addition, interferon mutant aggregation was repressed significantly by the addition of Tween-80, and a specific bioactivity of 7.35 x 10(8) +/- 0.56 x 10(8) IU mg(-1) was obtained. These results should be applicable to other low-stability recombinant proteins expressed in P. pastoris.
Wu
Dan
D
State Key Laboratory of Bioreactor Engineering, National Engineering Center of Biotechnology, East China University of Science and Technology, Shanghai, People's Republic of China.
Hao
Yu-You
YY
Chu
Ju
J
Zhuang
Ying-Ping
YP
Zhang
Si-Liang
SL
D019149:Bioreactors; D003470:Culture Media / Q000737:chemistry; D003902:Detergents / Q000494:pharmacology; D006801:Humans; D016898:Interferon-alpha / Q000235:genetics / Q000378:metabolism*; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism; D010843:Pichia / Q000378:metabolism*; D011136:Polysorbates / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
D003470:Culture Media; D003902:Detergents; D016898:Interferon-alpha; D050505:Mutant Proteins; D011136:Polysorbates; D011994:Recombinant Proteins; D010447:Peptide Hydrolases