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Electroinduced Extraction of Human Ferritin Heavy Chain Expressed in Hansenula polymorpha

Author:
Ganeva, Valentina, Galutzov, Bojidar, Angelova, Boyana, Suckow, Manfred
Source:
Applied biochemistry and biotechnology 2018 v.184 no.4 pp. 1286-1307
ISSN:
0273-2289
Subject:
Hansenula polymorpha, cell walls, ferritin, humans, iron, molecular weight, plasma membrane, porosity, pulsed electric fields, recombinant proteins
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.
Agid:
5927055