Yuyou Hao

Incomplete formation of intramolecular disulfide bond triggers degradation and aggregation of human consensus interferon-α mutant by Pichia pastoris

Previous study has shown that the degradation and aggregation of recombinant human consensus interferon-α 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 β-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.

Incomplete protein disulphide bond conformation and decreased protein expression result from high cell growth during heterologous protein expression in Pichia pastoris

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.