title: [Influence of methanol concentration on purification recovery of consensus interferon-a produced by Pichia pastoris].
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.
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Wu
authors.forename: Ju
authors.initials: J
authors.lastname: Chu
authors.forename: Yonghong
authors.initials: Y
authors.lastname: Wang
authors.forename: Yingping
authors.initials: Y
authors.lastname: Zhuang
authors.forename: Siliang
authors.initials: S
authors.lastname: Zhang
mesh_terms: D000998:Antiviral Agents / Q000302:isolation & purification; D005285:Fermentation; D006801:Humans; D016898:Interferon-alpha / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D000432:Methanol / Q000032:analysis*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
chemical_list: D000998:Antiviral Agents; D016898:Interferon-alpha; D011994:Recombinant Proteins; C050739:interferon alfacon-1; D000432:Methanol