Development of high cell density recombinant Pichia Pastoris culture for human growth hormone production
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Date
2008
Authors
Lee, Yoeu Chek
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Abstract
Methylotrophic yeast, Pichia pastoris has been used as an excellent host to
produce more than 400 recombinant heterologous proteins of academic and industrial
interests. Pichia pastoris strain used in this study was GS 115 2(5) with the
phenotype of Mut+, which has been genetically modified to produce intracellular
recombinant human growth hormone (rhGH). In this research, the development of
fermentation protocols for high cell density culture of recombinant Pichia pastoris
was carried out. Medium formulation and improved batch cultivation was
investigated prior to fed batch fermentation. Various feeding strategies and methanol
induction conditions on biomass and rHGH production were also studied. Ion
exchange chromatography was performed to purify the rhGH produced. A protocol
of the high cell density culture was developed which gave the highest dry cell weight
of 120 g/L with the maximum specific growth rate of 0.133 h-1. A protocol that
produces rHGH at the shortest time requirement was also obtained. It took only 39
hours from the inoculation to reach the rhGH production stage, which was almost
50% shorter time required compared to the preliminary 72 h. An enhanced induction
condition for rhGH production was also developed where a yield of 2.4 mg of rhGH
/L of lysate was achieved. A total of 349.5 μg of rhGH was purified from the total of
2418.8 μg of rhGH, which give the final yield of 14.5% and the purification factor of
1.5.
Description
Master
Keywords
Biological Science , Cell density , Hormone production