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Electroinduced Extraction of Human Ferritin Heavy Chain Expressed in Hansenula polymorpha

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Abstract

А protocol for the efficient and selective recovery of human ferritin heavy chain (FTH1) expressed intracellularly in Hansenula polymorpha was developed. It was based on electropermeabilisation and an increase in the cell wall porosity by pulsed electric field (PEF) treatment and subsequent incubation with a low concentration of a lytic enzyme. Irreversible plasma membrane permeabilisation was induced by applying rectangular electric pulses in the flow mode. The electrical treatment itself did not cause the release of the recombinant protein but induced the sensitisation of H. polymorpha cells to the lytic enzyme. Consequently, the subsequent incubation of the permeabilised cells with lyticase led to the recovery of approximately 90% of the recombinant protein, with a purification factor of 1.8. A similar efficiency was obtained by using the industrial lytic enzyme Glucanex. The released FTH1 appears in the form of an oligomer with a molecular mass of approximately 480 kDa, which is able to bind iron. The possibility for scaling the proposed protocol is discussed.

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Funding

This research received funding from the European Union Seventh Framework Programme (FP7/2012–2017) under grant agreement n° 312004.

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Authors and Affiliations

  1. Department Biophysics & Radiobiology, Biological Faculty, Sofia University, 8 Dragan Tzankov Blvd., 1164, Sofia, Bulgaria

    Valentina Ganeva, Bojidar Galutzov & Boyana Angelova

  2. ARTES Biotechnology GmbH, Elizabeth Selbert Str. 9, 40764, Langenfeld, Germany

    Manfred Suckow

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  1. Valentina Ganeva
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  2. Bojidar Galutzov
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  4. Manfred Suckow
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Correspondence to Valentina Ganeva.

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Highlights

• First evidence for electroinduced recovery of recombinant protein from H. polymorpha is presented.

• Protocol based on electropermeabilisation and subsequent incubation with lytic enzyme is proposed.

• The released FTH1 appears in the form of functionally active oligomer with a molecular mass of 480 kDa.

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Ganeva, V., Galutzov, B., Angelova, B. et al. Electroinduced Extraction of Human Ferritin Heavy Chain Expressed in Hansenula polymorpha . Appl Biochem Biotechnol 184, 1286–1307 (2018). https://doi.org/10.1007/s12010-017-2627-9

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  • DOI: https://doi.org/10.1007/s12010-017-2627-9

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