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Optimization of the fermentation and downstream processes for human enterokinase production in Pichia pastoris

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Abstract

Enterokinase is one of the most frequently used enzymes for the removal of affinity tags from target recombinant proteins. In this study, several fermentation strategies were assayed for the production of human enterokinase in Pichia pastoris under constitutive GAP promoter. Two of them with controlled specific growth rate during whole cultivation showed a very low enterokinase activity, under 1 U/ml, of the fermentation medium. On the contrary, the combined fermentation with a maximum specific growth rate at the initial phase of the fermentation and stationary-like phase during the rest of the fermentation showed a significant accumulation of the enterokinase in the medium, which counted up to 1400 U/ml. Lower cultivation temperature had a negative impact on the enzyme accumulation during this fermentation strategy. Downstream processes were focused on buffer environment optimization directly after cultivation, as at this time, the most amount of the activity is eliminated by endogenous proteases. Slightly positive effect on enzyme activity in the medium had an addition of liquid storage solution of EDTA and KOH to adjust pH to 8 and molarity of the EDTA to 50 mM. During the purification process, a significant amount of the enzyme was detected to be lost, which counted up to 90%. The purified enzyme, enterokinase, kept quality standard of the published enzymes.

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Acknowledgements

This work was supported by the Slovak Research and Development Agency under the contract No. APVV-0119-12.

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Authors and Affiliations

  1. Faculty of Natural Sciences, Department of Molecular Biology, Comenius University in Bratislava, Mlynská dolina, Ilkovičova 6, 842 15, Bratislava 4, Slovak Republic

    Kristína Melicherová, Ján Krahulec, Martin Šafránek, Veronika Lišková, Diana Hopková, Diana Széliová & Ján Turňa

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  1. Kristína Melicherová
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  2. Ján Krahulec
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  3. Martin Šafránek
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  4. Veronika Lišková
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  5. Diana Hopková
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  6. Diana Széliová
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  7. Ján Turňa
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Correspondence to Ján Krahulec.

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This work was supported by the Slovak Research and Development Agency under the contract No. APVV-0119-12.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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Melicherová, K., Krahulec, J., Šafránek, M. et al. Optimization of the fermentation and downstream processes for human enterokinase production in Pichia pastoris . Appl Microbiol Biotechnol 101, 1927–1934 (2017). https://doi.org/10.1007/s00253-016-7960-3

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  • DOI: https://doi.org/10.1007/s00253-016-7960-3

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