Glyco-engineered horseradish peroxidase for targeted cancer treatment / von Günther Bonifert
VerfasserBonifert, Günther
Begutachter / BegutachterinHerwig, Christoph ; Spadiut, Oliver
Umfang79 Bl. : Ill., graph. Darst.
HochschulschriftWien, Techn. Univ., Dipl.-Arb., 2015
Zsfassung in dt. Sprache
Schlagwörter (EN)HRP / peroxidase / glycosylation / glyco-engineering / Pichia pastoris / cancer treatment
URNurn:nbn:at:at-ubtuw:1-85034 Persistent Identifier (URN)
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Glyco-engineered horseradish peroxidase for targeted cancer treatment [1.57 mb]
Zusammenfassung (Englisch)

The oxidoreductase horseradish peroxidase (HRP; EC reacts with the plant hormone indole-3-acetic acid (IAA) to a cytotoxin and induces cell apoptosis. This feature can be used for targeted cancer therapy by transporting both the enzyme and the hormone to the cancer cells by antibody conjugation. Recombinant HRP, produced in the yeast Pichia pastoris, carries hypermannosylation on the enzyme surface. Due to this fact, the recombinant enzymes are not qualified for medical applications as there can be problems with antibody conjugation due to hyperglycosylation and furthermore these non-native extensive glycan chains can lead to immunogenic reactions in the patients. In this Thesis, 5 novel recombinant HRP variants were generated with reduced surface glycosylation. Two HRP variants described the isoenzyme HRP C1A but were expressed in glyco-engineered Pichia pastoris strains, whereas the other 3 variants described a mutated enzyme expressed as well in glyco-engineered Pichia pastoris strains. The enzyme with the most promising characteristics in terms of catalytic activity and stability was produced in the controlled environment of a bioreactor in batch and fed-batch mode. The purified and biochemically characterized enzymes (HRP with reduced glycosylation pattern, plant-HRP and wildtype-HRP for reference) were tested for their interactions and effects on cancer cell lines with IAA. The cytotoxicity studies revealed that the recombinant enzymes are a powerful tool and promising candidate for cancer treatment.