Incomplete formation of intramolecular disulfide bond triggers degradation and aggregation of human consensus interferon-alpha mutant by Pichia pastoris.
Previous study has shown that the degradation and aggregation of recombinant human consensus interferon-alpha mutant (cIFN) were serious when cIFN was secreted to bioreactor by Pichia pastoris. In this study, we showed that this phenomenon was concomitant well with the formation of the doublets of cIFN monomers that could be seen clearly on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The doublets were a mixture of two isomers formed by cIFN with different disulfide bonds and identified that the upper cIFN in doublets contains only one disulfide bond while the lower cIFN contains intact disulfide bonds by a novel method termed protein laddering map on SDS-PAGE. In addition, the instability of cIFN with different disulfide bond forms is also analyzed through a novel in vitro conversion assay based on incubation with different concentrations of beta-mercaptoethanol. The results showed that only a wound such as cleavage of only one disulfide bond could be fatal to cIFN stability. If the disulfide bonds in cIFN monomers were broken, three kinds of aggregates would be formed easily: covalent aggregates, non-covalent aggregates, and unknown dimers. Likewise, the unfolded species also displayed reduced stability to proteolysis. These results indicate that the incomplete formation of disulfide bond in cIFN secreted to fermentation broth triggers severe degradation and aggregation of cIFN, which result in sharp decrease of bioactivity of cIFN in bioreactor.
Wu
Dan
D
State Key Laboratory of Bioreactor Engineering, National Engineering Center of Biotechnology, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People's Republic of China.
Ma
Dong
D
Hao
Yu-You
YY
Chu
Ju
J
Wang
Yong-Hong
YH
Zhuang
Ying-Ping
YP
Zhang
Si-Liang
SL
D019149:Bioreactors; D004220:Disulfides / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D005285:Fermentation; D006801:Humans; D016898:Interferon-alpha / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010843:Pichia / Q000235:genetics / Q000254:growth & development*; D055550:Protein Stability; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
D004220:Disulfides; D016898:Interferon-alpha; D011994:Recombinant Proteins