Date

2018

Author

Akgün, Yiğit

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The objectives of this thesis are investigation of the effects of oxygen transfer conditions on the recombinant human growth hormone (rhGH) production by Pichia pastoris under glyceraldehyde-3-phosphate dehydrogenase promoter (PGAP); and, separation and purification of the synthesized and secreted rhGH by zeolitic imidazolate framework-8 adsorbent (ZIF-8). The effects of the oxygen transfer were studied at three conditions at constant dissolved oxygen concentrations (CDO) of CDO= 1%, 5%, and 15%. The highest cell and rhGH concentrations were obtained at CDO = 15% condition, respectively as, 177.3 g/L and 40.5 mg/L. The cell concentration at CDO= 15% condition was 1.7- and 3.4-fold higher; whereas, CrhGH was 2.04- and 2.64-fold higher, than those obtained at CDO = 5% and CDO = 1% conditions, respectively. For the separation and purification of rhGH, ZIF-8 was synthesized. Besides rhGH, recombinant granulocyte colony-stimulating factor (rGCSF) was used as the second model protein to be separated for comparison. Although ZIF-8 has high adsorption performance, was not selective in the separation of the r-proteins. Thus, metal and ligand addition-based modifications, such as nickel impregnation, bimetallic Ni/ZIF-8 syntheses, addition of a terminating ligand (1-methylimidazole), and surface ligand exchange reaction with histamine dihydrochloride, were applied separately. Amongst, the last two were used to create a specific molecular-separation-arm (spacer) in ZIF-8. ZIF-8 was synthesized by the solvothermal/hydrothermal method with a yield of 37±0.5 %. ZIF-8 synthesis yield decreased to 9±0.5 % by the addition of 1-methylimidazole (1-MI) into the reaction mixture. NaOH was used to increase the syntheses yields of ZIF-8 and 1-MI-ZIF-8; thus, ZIF-8 and 1-MI-ZIF-8 were synthesized, respectively, with 99±0.5 % and 41±0.5 % yields. The highest protein adsorption attained at pH = 5.0, and the elution at T = 35 ˚C were determined as the optimum separation conditions. Although the eluents glycerol and L-histidine showed better performance than those of NaCl, NTA, Na2HPO4, and MgCl2.6H2O; they also did not provide a selective separation and purification. Thus, ZIF-8 needs to be modified with further modifications to create a selective resin for the IMAC process.  

Subject Keywords

Somatotropin., Growth hormone releasing factor., Pichia pastoris., Protein engineering., Biochemical engineering., Metabolism

URI

http://etd.lib.metu.edu.tr/upload/12621806/index.pdf
https://hdl.handle.net/11511/27076

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Graduate School of Natural and Applied Sciences, Thesis