<div class="csl-bib-body">
<div class="csl-entry">Ebner, J. (2021). <i>Development of a refolding and capture protocol for HRP IBs produced in E. coli</i> [Diploma Thesis, Technische Universität Wien]. reposiTUm. https://doi.org/10.34726/hss.2021.87273</div>
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dc.identifier.uri
https://doi.org/10.34726/hss.2021.87273
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dc.identifier.uri
http://hdl.handle.net/20.500.12708/16779
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dc.description.abstract
While Horseradish Peroxidase (HRP) is an already established enzyme used in diagnostic kits and immunoassays, it saw a rising interest in the past years due to the possible application in targeted cancer treatment. This requires a reliable supply of well-defined HRP. However, it is currently only produced from its natural source, the horseradish root, where it occurs in at least 15 different isoforms and exhibits an inhomogeneous plant glycosylation pattern. While several of studies have reported the recombinant production in a variety of hosts, none of them have presented a viable alternative to current production, mainly due to low yields. One organism that can potentially produce HRP up to high titers is E. coli. In this case, though, HRP is not produced in its active form, but as Inclusion bodies (IBs). Refolding yields of HRP IBs are low, mitigating the high titers achieved during the fermentation.In this work, the unit operations solubilization, refolding and capture were investigated in order to obtain active HRP from HRP IBs. Three distinct sections are presented here, they are, however, not equivalent to the unit operations. Instead, for the first section, the unit operations solubilization and refolding were investigated in an integrated approach using a small-scale Design of Experiment approach. The main focus was put on the redox conditions during solubilization and refolding, which enables the formation of disulfide bonds and proved to be essential for the correct folding of HRP. For the second section, a scale-up to a refolding reactor was performed. This allowed for a controlled environment and monitoring of the redox level during refolding. In addition, a fed batch refolding approach was tested in order to enhance refolding yields. With the tried methods, however, no improvement of the refolding yield could be achieved in comparison to the batch refolding mode. For the third section, a suitable capture step using salt precipitation of impurities followed by a hydrophobic interaction chromatography was investigated.Based on the results of these three parts, a process which resulted in a specific activity of 980 U/mg and a purity of 98% according to SEC-HPLC could be established. Furthermore, a yield of 567 mg active HRP / L fermentation broth could be achieved. Additionally, the advantages of an integrated approach, especially for solubilization and refolding, could be shown in this work.
en
dc.language
English
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dc.language.iso
en
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dc.rights.uri
http://rightsstatements.org/vocab/InC/1.0/
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dc.subject
Protein Refolding
en
dc.subject
Horseradish peroxidase
en
dc.subject
Inclusion body processing
en
dc.subject
Capture and Concentration
en
dc.subject
Fed-Batch refolding
en
dc.title
Development of a refolding and capture protocol for HRP IBs produced in E. coli
en
dc.type
Thesis
en
dc.type
Hochschulschrift
de
dc.rights.license
In Copyright
en
dc.rights.license
Urheberrechtsschutz
de
dc.identifier.doi
10.34726/hss.2021.87273
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dc.contributor.affiliation
TU Wien, Österreich
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dc.rights.holder
Julian Ebner
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dc.publisher.place
Wien
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tuw.version
vor
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tuw.thesisinformation
Technische Universität Wien
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dc.contributor.assistant
Humer, Diana Claudia
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tuw.publication.orgunit
E166 - Institut für Verfahrenstechnik, Umwelttechnik und technische Biowissenschaften
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dc.type.qualificationlevel
Diploma
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dc.identifier.libraryid
AC16131954
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dc.description.numberOfPages
52
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dc.thesistype
Diplomarbeit
de
dc.thesistype
Diploma Thesis
en
dc.rights.identifier
In Copyright
en
dc.rights.identifier
Urheberrechtsschutz
de
tuw.advisor.staffStatus
staff
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tuw.assistant.staffStatus
staff
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item.openaccessfulltext
Open Access
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item.openairecristype
http://purl.org/coar/resource_type/c_bdcc
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item.grantfulltext
open
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item.mimetype
application/pdf
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item.languageiso639-1
en
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item.openairetype
master thesis
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item.fulltext
with Fulltext
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item.cerifentitytype
Publications
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crisitem.author.dept
E058-05-2 - Fachgruppe Förderberatung - angewandte Forschung und Wirtschaftskooperation
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crisitem.author.parentorg
E058-05 - Fachbereich Förderberatung und Wirtschaftskooperationen