Graduate theses
Current Record: 20 of 233
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Title |
Δημιουργία στελεχών Pichia pastoris για την ετερόλογη έκφραση πρωτεϊνών γλυκιάς γεύσης |
Author
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Βουγιατζή, Αικατερίνη Σταυρούλα
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Thesis advisor
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Παυλίδης, Ιωάννης
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Abstract |
One of the most popular heterologous protein expression systems is the methylotrophic
yeast Pichia pastoris. Using the yeast metabolic pathway for the utilization of methanol
(Methanol Utilization pathway, MUt), and specifically the characteristic of the alcohol
oxidase (AOX) promoter being activated only when methanol is present in the
environment of the cells, the tightly controlled expression of heterologous proteins can
take place. Being a eukaryotic microorganism, P. pastoris offers a more complex protein
expression system than bacterial hosts, such as E. coli, while being a yeast, it is a cheaper
and less time-consuming system than mammalian cells.
The objective of this project was the preparation of Pichia pastoris strains for the
heterologous expression of five hypothetical and two known sweet proteins. The first
step of the process was to transform the yeast strain GS115 with linearized plasmid
vector pPIC9K. In the following steps, growth experiments were performed on the
transformed cells under specific conditions in solid-state cultures. The MUts or MUt+
phenotype of the colonies was determined, as well as their genotype by detecting the
desired genes in the yeast genome using polymerase chain reaction (PCR). Finally,
cultures were prepared for the expression of the proteins of interest and affinity
chromatography was used for their purification. The expression of the two proteins Mab
II and MNEI was observed through the electrophoresis under denaturing conditions
(SDS-PAGE), but in low yields.
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Language |
Greek |
Issue date |
2022-11-24 |
Collection
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School/Department--School of Sciences and Engineering--Department of Chemistry--Graduate theses
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Type of Work--Graduate theses
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Permanent Link |
https://elocus.lib.uoc.gr//dlib/7/d/6/metadata-dlib-1667896608-517701-9912.tkl
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Views |
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