1. ZHAW digitalcollection
  2. Life Sciences und Facility Management
  3. Publikationen
  4. Publikationen Life Sciences und Facility Management
Please use this identifier to cite or link to this item: https://doi.org/10.21256/zhaw-25703
Full metadata record
DC FieldValueLanguage
dc.contributor.authorRaschmanová, Hana-
dc.contributor.authorPaulová, Leona-
dc.contributor.authorBranská, Barbora-
dc.contributor.authorKnejzlík, Zdeněk-
dc.contributor.authorMelzoch, Karel-
dc.contributor.authorKovar, Karin-
dc.date.accessioned2022-09-30T09:30:35Z-
dc.date.available2022-09-30T09:30:35Z-
dc.date.issued2018-11-
dc.identifier.issn0015-5632de_CH
dc.identifier.issn1874-9356de_CH
dc.identifier.urihttps://digitalcollection.zhaw.ch/handle/11475/25703-
dc.descriptionErworben im Rahmen der Schweizer Nationallizenzen (http://www.nationallizenzen.ch)de_CH
dc.description.abstractPharmaceutical grade trypsin is in ever-increasing demand for medical and industrial applications. Improving the efficiency of existing biotechnological manufacturing processes is therefore paramount. When produced biotechnologically, trypsinogen-the inactive precursor of trypsin-is advantageous, since active trypsin would impair cell viability. To study factors affecting cell physiology and the production of trypsinogen in fed-batch cultures, we built a fusion protein of porcine trypsinogen and enhanced green fluorescent protein (EGFP) in Pichia pastoris. The experiments were performed with two different pH values (5.0 and 5.9) and two constant specific growth rates (0.02 and 0.04 1/h), maintained using exponential addition of methanol. All the productivity data presented rely on an active determination of trypsin obtained by proteolysis of the trypsinogen produced. The pH of the medium did not affect cell growth, but significantly influenced specific production of trypsinogen: A 1.7-fold higher concentration of trypsinogen was achieved at pH 5.9 (64 mg/L at 0.02 1/h) compared to pH 5.0. EGFP was primarily used to facilitate detection of intracellular protein over the biosynthetic time course. Using flow cytometry with fluorescence detection, cell disruption was avoided, and protein extraction and purification prior to analysis were unnecessary. However, Western blot and SDS-PAGE showed that cleavage of EGFP-trypsinogen fusion protein occurred, probably caused by Pichia-endogenous proteases. The fluorescence analysis did therefore not accurately represent the actual trypsinogen concentration. However, we gained new experimentally-relevant insights, which can be used to avoid misinterpretation of tracking and quantifying as well as online-monitoring of proteins with the frequently used fluorescent tags.de_CH
dc.language.isoende_CH
dc.publisherSpringerde_CH
dc.relation.ispartofFolia Microbiologicade_CH
dc.rightsLicence according to publishing contractde_CH
dc.subjectAnimalde_CH
dc.subjectCulture mediade_CH
dc.subjectGene expressionde_CH
dc.subjectGreen fluorescent proteinde_CH
dc.subjectHydrogen-ion concentrationde_CH
dc.subjectPichiade_CH
dc.subjectProtein processing, post-translationalde_CH
dc.subjectRecombinant fusion proteinde_CH
dc.subjectSwinede_CH
dc.subjectTrypsinogende_CH
dc.subject.ddc660.6: Biotechnologiede_CH
dc.titleProduction and cleavage of a fusion protein of porcine trypsinogen and enhanced green fluorescent protein (EGFP) in Pichia pastorisde_CH
dc.typeBeitrag in wissenschaftlicher Zeitschriftde_CH
dcterms.typeTextde_CH
zhaw.departementLife Sciences und Facility Managementde_CH
zhaw.organisationalunitInstitut für Chemie und Biotechnologie (ICBT)de_CH
dc.identifier.doi10.1007/s12223-018-0619-yde_CH
dc.identifier.doi10.21256/zhaw-25703-
dc.identifier.pmid29872953de_CH
zhaw.funding.euNode_CH
zhaw.issue6de_CH
zhaw.originated.zhawYesde_CH
zhaw.pages.end787de_CH
zhaw.pages.start773de_CH
zhaw.publication.statuspublishedVersionde_CH
zhaw.volume63de_CH
zhaw.publication.reviewPeer review (Publikation)de_CH
zhaw.author.additionalNode_CH
zhaw.display.portraitYesde_CH
Appears in collections:Publikationen Life Sciences und Facility Management

Files in This Item:
File Description SizeFormat 
2018_Raschmanova-etal_Fusion-protein-of-porcine-trypsinogen-and-EGFP-Pichia-pastoris.pdf1.19 MBAdobe PDFThumbnail
View/Open
Show simple item record
Raschmanová, H., Paulová, L., Branská, B., Knejzlík, Z., Melzoch, K., & Kovar, K. (2018). Production and cleavage of a fusion protein of porcine trypsinogen and enhanced green fluorescent protein (EGFP) in Pichia pastoris. Folia Microbiologica, 63(6), 773–787. https://doi.org/10.1007/s12223-018-0619-y
Raschmanová, H. et al. (2018) ‘Production and cleavage of a fusion protein of porcine trypsinogen and enhanced green fluorescent protein (EGFP) in Pichia pastoris’, Folia Microbiologica, 63(6), pp. 773–787. Available at: https://doi.org/10.1007/s12223-018-0619-y.
H. Raschmanová, L. Paulová, B. Branská, Z. Knejzlík, K. Melzoch, and K. Kovar, “Production and cleavage of a fusion protein of porcine trypsinogen and enhanced green fluorescent protein (EGFP) in Pichia pastoris,” Folia Microbiologica, vol. 63, no. 6, pp. 773–787, Nov. 2018, doi: 10.1007/s12223-018-0619-y.
RASCHMANOVÁ, Hana, Leona PAULOVÁ, Barbora BRANSKÁ, Zdeněk KNEJZLÍK, Karel MELZOCH und Karin KOVAR, 2018. Production and cleavage of a fusion protein of porcine trypsinogen and enhanced green fluorescent protein (EGFP) in Pichia pastoris. Folia Microbiologica. November 2018. Bd. 63, Nr. 6, S. 773–787. DOI 10.1007/s12223-018-0619-y
Raschmanová, Hana, Leona Paulová, Barbora Branská, Zdeněk Knejzlík, Karel Melzoch, and Karin Kovar. 2018. “Production and Cleavage of a Fusion Protein of Porcine Trypsinogen and Enhanced Green Fluorescent Protein (EGFP) in Pichia Pastoris.” Folia Microbiologica 63 (6): 773–87. https://doi.org/10.1007/s12223-018-0619-y.
Raschmanová, Hana, et al. “Production and Cleavage of a Fusion Protein of Porcine Trypsinogen and Enhanced Green Fluorescent Protein (EGFP) in Pichia Pastoris.” Folia Microbiologica, vol. 63, no. 6, Nov. 2018, pp. 773–87, https://doi.org/10.1007/s12223-018-0619-y.


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.