METHOD DEVELOPMENT IN ASSESSING RECOMBINANT HUMAN GROWTH HORMONE EXPRESSION FROM Pichia pastoris
| dc.contributor.author | CHAI, HUI LING | |
| dc.date.accessioned | 2016-01-12T03:28:08Z | |
| dc.date.available | 2016-01-12T03:28:08Z | |
| dc.date.issued | 2007 | |
| dc.description.abstract | Recombinant human growth hormone (rhGH) was expressed in genetically modified Pichia pastoris strain GS 115 2(5) in a two-stage cultivation process. However, the preliminary cultivation process and purification strategies of rhGH from strain GS115 2(5) were still an open issue. The main aim of this research is to evaluate the downstream processes specifically to quantify and purify the pharmaceutical products. The culture was first grown in a glycerol-containing production medium as the sole carbon source prior to obtain high cell biomass and was transferred to the second stage where rhGH was induced with methanol as the sole inducer in the medium. rhGH expression from the cultures was confirmed with gel electrophoresis, Western blotting and ELISA. Different cell disruption methods such as glass beads, French press, sonicator, modified protein extraction fluid and YPES were evaluated in terms of efficiency. Fermentation process using methanol as sole carbon source from 0 hour to 204 hours was performed to determine the optimal induction period of the rhGH expression by using the optimized bead milling method. The profile of cell dry weight and the rhGH expression was maintained constantly at 58 gIL and 330 Ilg/L. Also, the effect of methanol concentration and flask design on the growth of P. pastoris and rhGH expression were investigated. The biopharmaceutical compound was purified with chromatographic methods and passive diffusion from polyacrylamide gel, with an overall recovery yield of 26.4%. Isoelectric point of rhGH at pI 5.3 was determined with isoelectric focusing gel. Low expression level of rhGH has prompted the detection of protease by using zymogram | en_US |
| dc.identifier.uri | http://hdl.handle.net/123456789/1376 | |
| dc.subject | RECOMBINANT HUMAN | en_US |
| dc.subject | GROWTH HORMONE | en_US |
| dc.title | METHOD DEVELOPMENT IN ASSESSING RECOMBINANT HUMAN GROWTH HORMONE EXPRESSION FROM Pichia pastoris | en_US |
| dc.type | Thesis | en_US |
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