Analysis and characterization of the pichia pastoris secretome for pharmaceutical protein production

Kırlı, Hatice Pınar
Human growth hormone (hGH) is used for the treatment of many diseases like short stature in children. It is generally purified by using affinity chromatography. Due to the high cost of affinity chromatography, other chromatographic techniques have been investigated for purification of recombinant human growth hormone (rhGH). The aim of this work was to explore chromatographic purification protocols for rhGH from the secretome of Pichia pastoris. First, the entire secretome was examined by 2-D gel electrophoresis. A major 4 unique proteins apart from rhGH were found in the secretome. The molecular weights of the other proteins were found to range between 20 and 140 kDa. The isoelectric points of most of the proteins in secretome were determined to vary between 4.4 and 5.7. To putatively identify the proteins in the secretome, the secretome of P. pastoris from the literature was analyzed using the software JVirGel 2.0, which forms a virtual 2D gel image. By comparing the virtual image with the experimental gel results, the possible identities for the experimental bands were suggested as the paf1 complex component, cell wall protein DAN4, protein phosphatase, lectin-like protein, putative glucanases, protein kinase, aspartic proteinase 3, repressible acid phosphatase. After the characterization of the secretome proteins, the purification of rhGH was investigated using desalting, size exclusion and anion exchange chromatography. In the desalting column, proteins were separated from impurities like salts and peptide parts. It was found that using size exclusion chromatography, the rhGH was partially purified from proteins with molecular weight lower than 15 kDa. It was predicted that rhGH interacts with other proteins and forms agglomerates. Although partial purification of rhGH is possible by using consecutive usage of two chromatographic techniques, the separation yield is low, since sequential chromatographic techniques probably caused substantial protein loss. As the final investigation of this work, secretomes produced by two different promoters were compared for purification of rhGH. Fractions of size exclusion and anion exchange chromatography were analyzed by SDS-PAGE. It was found that the secretomes were mostly similar except for the fact that the rhGH concentration of the secretome with the modified GAP promoter was higher than that for the secretome with modified AOX. In conclusion, rhGH was partially purified by using size exclusion and anion exchange chromatography.


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Citation Formats
H. P. Kırlı, “Analysis and characterization of the pichia pastoris secretome for pharmaceutical protein production,” Thesis (M.S.) -- Graduate School of Natural and Applied Sciences. Chemical Engineering., Middle East Technical University, 2019.