Effect of glucose frucrose mixed feeding on human growth hormone production under GAP promoter by Pichia pastoris

Date

2016-04-06

Author

Duygu, Yalçınkaya
Hoxha, Bebeta
Çalık, Pınar

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Pichia pastorishas become one of the most preferable eukaryotic host microorganismswith its success in expression of heterologous proteins. Glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP) has become prominent with its strong and constitutive expression. Carbon source selection is an important factor on growth and expression in GAP-driven systems.Glycerol and glucose are usedmostly assole carbon sourcesfor the fed-batch process. However other sugars like fructose, sorbitol, and mannitol can be used as additional carbons since they are also utilized in the carbon metabolism in the glycolytic pathway.Human growth hormone (hGH)is a non-glycosylatedprotein that has191 amino acid residues in its structure with a molecular mass of 22 kDa. The aim of this study was to compare the feeding media for fed-batch process to increase the production of hGH.Firstly, we ligated His-tagged hGH gene to pGAPZαA plasmid with alpha factor as secretion signal. The plasmid, subsequently, wastransformed into wild type X-33 Pichia pastoris. By using Dot-Blot analysis the best producing strain was selected. Cell growing and extracellular proteinexpression were studied by using pilot scale bioreactor when glucose and glucose:fructose mixturewere used as carbon sources. Glucose and glucose:fructose mixture werefed to the medium exponentially. Two different predetermined specific growth rates were applied μ=0.1h-1and μ=0.07h-1. The results were compared with experiments where glucose was used as the carbonsource.A higher concentration of hGH was obtained by using glucose:fructosemixture as substrate. The fermentation characteristics will be presented in details.

URI

https://hdl.handle.net/11511/86996

Conference Name

Pichia 2016 Conference (3 - 06 Nisan 2016)

Collections

Department of Chemical Engineering, Conference / Seminar

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Citation Formats

Y. Duygu, B. Hoxha, and P. Çalık, “Effect of glucose frucrose mixed feeding on human growth hormone production under GAP promoter by Pichia pastoris,” presented at the Pichia 2016 Conference (3 - 06 Nisan 2016), 2016, Accessed: 00, 2021. [Online]. Available: https://hdl.handle.net/11511/86996.