title: Incomplete protein disulphide bond conformation and decreased protein expression result from high cell growth during heterologous protein expression in Pichia pastoris.
abstract: Previous report has shown that the expression of recombinant human consensus interferon-α mutant (cIFN) in Pichia pastoris in bioreactor is limited with respect to the incorrectly folded cIFN with incomplete disulfide bond, which lead to the degradation and aggregation of cIFN. In this study, the origin of incorrectly folded cIFN is firstly studied. Fed-batch fermentation in bioreactor shows that the incorrectly folded cIFN is formed intramolecularly and secreted to the extracellular environment. Further chemostat cultures indicate that the specific growth rate is the critical factor for the production of incorrect cIFN. In addition, cell shows reduced expression level of cIFN at high specific growth rate. We also demonstrate that the incorrectly folded cIFN could form aggregates intracellularly and these aggregates are non-covalent forms. Taken together, these results suggest that the efficient heterologous expression of cIFN is limited by high cell growth that is unique from expression limitations seen for soluble proteins. A balance has to be found between the increase for high efficient expression of heterologous proteins and requirement of the high cell growth during the expression of recombinant proteins in P. pastoris.
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Ave, Wuxi, Jiangsu 214122, People's Republic of China.
authors.forename: Dan
authors.initials: D
authors.lastname: Wu
authors.forename: Ju
authors.initials: J
authors.lastname: Chu
authors.forename: Yu-You
authors.initials: YY
authors.lastname: Hao
authors.forename: Yong-Hong
authors.initials: YH
authors.lastname: Wang
authors.forename: Ying-Ping
authors.initials: YP
authors.lastname: Zhuang
authors.forename: Si-Liang
authors.initials: SL
authors.lastname: Zhang
mesh_terms: D015153:Blotting, Western; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism*; D005285:Fermentation; D006801:Humans; D016898:Interferon-alpha / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D010843:Pichia / Q000254:growth & development* / Q000378:metabolism*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism
chemical_list: D004220:Disulfides; D016898:Interferon-alpha; D011994:Recombinant Proteins; C050739:interferon alfacon-1