Kinetic studies of constitutive human granulocyte-macrophage colony stimulating factor (hGM-CSF) expression in continuous culture of Pichia pastoris.
Extracellular human granulocyte-macrophage colony stimulating factor (hGM-CSF) expression was studied under the control of the GAP promoter in recombinant Pichia pastoris in a series of continuous culture runs (dilution rates from 0.025 to 0.2 h(-1)). The inlet feed concentration was also varied and the steady state biomass concentration increased proportionally demonstrating efficient substrate utilization and constancy of the biomass yield coefficient (Y(x/s)) for a given dilution rate. The specific product formation rate (q(P)) showed a strong correlation with dilution rates demonstrating growth associated product formation of hGM-CSF. The volumetric product concentration achieved at the highest feed concentration (4x) and a dilution rate of 0.2 h(-1) was 82 mg l(-1) which was 5-fold higher compared to the continuous culture run with 1x feed concentration at the lowest dilution rate thus translating to a 40 fold increase in the volumetric productivity. The specific product yield (Y(P/X)) increased slightly from 2 to 2.5 mg g(-1), with increasing dilution rates, while it remained fairly invariant, for all feed concentrations demonstrating negligible product degradation or feed back inhibition. The robust nature of this expression system would make it easily amenable to scale up for industrial production.
Khasa
Yogender Pal
YP
School of Biotechnology, Jawaharlal Nehru University, New Delhi, 110067, India.
Khushoo
Amardeep
A
Srivastava
Leena
L
Mukherjee
K J
KJ
D018533:Biomass; D003470:Culture Media; D046508:Culture Techniques / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010843:Pichia / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*
D003470:Culture Media; D016178:Granulocyte-Macrophage Colony-Stimulating Factor