title: Enhancing toxic protein expression in Escherichia coli fed-batch culture using kinetic parameters: Human granulocyte-macrophage colony-stimulating factor as a model system.
abstract: The kinetics of recombinant human granulocyte-macrophage colony-stimulating factor (hGM-CSF) expression was studied under the strong T7 promoter in continuous culture of Escherichia coli using complex medium to design an optimum feeding strategy for high cell density cultivation. Continuous culture studies were done at different dilution rates and the growth and product formation profiles were monitored post-induction. Recombinant protein expression was in the form of inclusion bodies with a maximum specific product formation rate (q(p)) of 63.5 mg g(-1) DCW h(-1) at a dilution rate (D) of 0.3 h(-1). The maximum volumetric product concentration achieved at this dilution rate was 474 mg l(-1), which translated a ~1.4 and ~1.75 folds increase than the values obtained at dilution rates of 0.2 h(-1) and 0.4 h(-1) respectively. The specific product yield (Y(P/x)) peaked at 138 mg g(-1) DCW, demonstrating a ~1.6 folds increase in the values obtained at other dilution rates. A drop in q(p) was observed within 5-6 h of induction at all the dilution rates, possibly due to protein toxicity and metabolic stress associated with protein expression. The data from the continuous culture studies allowed us to design an optimal feeding strategy and induction time in fed-batch cultures which resulted in a maximum product concentration of 3.95 g l(-1) with a specific hGM-CSF yield (Y(P/x)) of 107 mg g(-1) DCW.
authors.affiliation: Department of Microbiology, University of Delhi South Campus, New Delhi, India. yogi110@gmail.com
authors.forename: Yogender Pal
authors.initials: YP
authors.lastname: Khasa
authors.forename: Amardeep
authors.initials: A
authors.lastname: Khushoo
authors.forename: Krishna Jyoti
authors.initials: KJ
authors.lastname: Mukherjee
mesh_terms: D061249:Batch Cell Culture Techniques; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism; D007700:Kinetics; D008954:Models, Biological; D011994:Recombinant Proteins / Q000096:biosynthesis
chemical_list: D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor