Pakistan Research Repository

Production of Recombinant Ptoteins Using Pichia Pastoris As An Expression Host

Adnan, Dr. Ahmed (2007) Production of Recombinant Ptoteins Using Pichia Pastoris As An Expression Host. Final Report: HEC Post Doc Fellowship Phase II. (Submitted)

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Abstract

The present study is concerned with the production of recombinent protein biopharmaceuticals such as Hepatitis B surface antigen (HBsAg) and Insulin Precursor from different clones of the methylotrophic yeast Pichia pastoris. Clones had already been prepared and are available at Helmholtz Zentrum für Infektionsforschung. Kinetic behavior and the productivity of Pichia pastoris HBsAg 1-copy and Pichia pastoris HBsAg 8-copy clone were investigated in oxygen limited fed batch cultures using 10 L stirred tank bioreactors. The glycerol was used as the carbon and energy source for the growth of the yeast in the fermenter but the yeast produced recombinant proteins only when it was induced and the glycerol was replaced with methanol. Promising results were obtained with one copy and eight copy clones. Although very high cell density could be obtained in case of one copy clone but the concentration of HBsAg was highest in case of the eight copy clone. So the eight copy clone was selected for futher studies. It was found that maximum concentration (2 grams/ L) of HBsAg could be obtained after 120 hours of induction. Samples from the bioreacter were also subjected to proteomic analysis. Changes in the proteomic spectrum in different phases of fermentation, were investigated by 2-D gel electrophoresis and MALDI ToF MS analysis. 2-D gel electrophoresis has shown many previously unidentified proteins, some of which are only appearing after induction. The clone ‘Pichia pastoris Insulin Precursor’ was used to invetigate the production of a mini-proinsulin precursor in high cell density cultivations using the 15 L stirred fermenter. The production was studied in oxygen limited fed-batch cultures. The cells secreted the proinsulin into the medium and its concentration after 72 hours of induction was approx. 2-3 g/L Pichia pastoris D2B was also investigated for the production of Dengue tetravalent vaccine in in shake flask cultures but the expression was very low. So it was not studied further in bioreactor cultures.

Item Type:Other
Additional Information:Helmholtz Zentrum fur Infektionsforschung Inhoffenstrabe
Subjects:Biological & Medical Sciences (c)
ID Code:3528
Deposited By:Mr. Javed Memon
Deposited On:30 Jun 2010 15:10
Last Modified:30 Jun 2010 15:10

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