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Combined effect of gene dosage and process optimization strategies on high-level production of recombinant human interleukin-3 (hIL-3) in Pichia pastoris fed-batch culture

Author:
Dagar, Vikas Kumar, Khasa, Yogender Pal
Source:
International journal of biological macromolecules 2018 v.108 pp. 999-1009
ISSN:
0141-8130
Subject:
Pichia pastoris, batch fermentation, bioactive properties, culture flasks, gene dosage, genome, humans, interleukin-3, quantitative polymerase chain reaction
Abstract:
In this work, the combined effects of gene dosage and process optimization strategies were studied to achieve higher hIL-3 expression in Pichia system. The in-vitro multimerization method was used to generate various Pichia X-33 transformants having multi-copy expression cassettes. The quantitative polymerase chain reaction (qPCR) strategy was used to further confirm the genome integration of hIL-3 expression cassette. From shake flask expression studies, the recombinant hIL-3 concentration in culture supernatant increased upto 8 copies to a level of 310mg/L, thereafter a considerably lower expression was observed. The small scale optimization experiments at shake flask level resulted in an improved product concentration of 350mg/L. The batch and fed-batch fermentation runs in complex medium showed a product concentration of 1.81 and 1.49g/L, respectively. To further enhance the production level, the fermentation runs were conducted in modified minimal media where a maximum hIL-3 protein level of 2.23g/L was obtained in batch fermentation. The specific product yield (YP/X) was at a level of 25.65mg/g DCW, whereas the overall volumetric productivity of the process was 27.31mg/L/h. The biological activity of the partially purified hIL-3 protein was confirmed via the proliferation of human erythroleukemia TF-1 cells using MTT assay.
Agid:
5857937