Influence of methanol concentration on purification recovery of consensus interferon-a produced by Pichia pastoris.
- Author:
Dan WU
1
;
Ju CHU
;
Yonghong WANG
;
Yingping ZHUANG
;
Siliang ZHANG
Author Information
1. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
- Publication Type:Journal Article
- MeSH:
Antiviral Agents;
isolation & purification;
Fermentation;
Humans;
Interferon-alpha;
biosynthesis;
genetics;
isolation & purification;
Methanol;
analysis;
Pichia;
genetics;
metabolism;
Recombinant Proteins;
biosynthesis;
genetics;
isolation & purification
- From:
Chinese Journal of Biotechnology
2011;27(12):1789-1796
- CountryChina
- Language:Chinese
-
Abstract:
We observed the influence of methanol concentration on purification recovery of recombinant human consensus interferon-a (cIFN) produced by Pichia pastoris. Fermentations controlled at 0.75% (W/V) methanol showed a 24% increase in clFN expression compared to using 0.25% methanol. However, the purification recovery rate of cIFN at 0.25% methanol was 3.75-fold higher than that at 0.75% methanol. To seek the reason, we analyzed the stability of clFN by SDS-PAGE and Native-PAGE as well as Western blotting. The electrophoresis results revealed that cIFN formed a lot of aggregates in media when the induction was controlled at 0.75% methanol, and two different aggregate forms were found: disulfide bond covalent aggregates and non-covalent aggregates. However, these aggregates almost disappeared when the methanol concentration was controlled at 0.25%, at the same time, cIFN bioactivity of supernatant increased almost 4.48-fold. Finally, 0.73g monomer cIFN was obtained after purification from 1 liter supernatant at 0.25% methanol induction, showed a 2.84-fold increase compare to the induction at 0.75% methanol.
