Kinetic studies of recombinant human interferon-gamma expression in continuous cultures of E. coli.
A series of continuous cultures was performed to understand the product formation kinetics of recombinant human interferon gamma (rhIFN-gamma) in Escherichia coli at different dilution rates ranging from 0.1 to 0.3 h(-1) in different media. A T7 promoter-based vector was used for expression of IFN-gamma in E. coli BL21 (DE3) cells. The recombinant protein was produced as inclusion bodies, thus allowing a rapid buildup of rhIFN-gamma inside the cell, with the specific product yield (Y(p/X)) reaching a maximum value of 182 mg g(-1) dry cell weight (DCW). In all the media tested, the specific product formation rate (q(p)) was found to be strongly correlated with the specific growth rate (mu), demonstrating the growth-associated nature of product formation. The q(p) values show no significant decline with time postinduction, even though the recombinant protein has been over produced inside the cell. The maximum q(p) level of 75.5 mg g(-1) h(-1) was achieved at the first hour of induction at the dilution rate of 0.3 h(-1). Also, this correlation between q(p) and mu was not critically dependent on media composition, which would made it possible to grow cells in defined media in the growth phase and then push up the specific growth rate just before induction by pulse addition of glucose and yeast extract. This would ensure the twin objectives of high biomass and high specific productivities, leading to high volumetric product concentration.
Vaiphei
S Thangminlal
ST
School of Biotechnology, Jawaharlal Nehru University, New Delhi, India. vaiphest@umdnj.edu
Pandey
Gaurav
G
Mukherjee
K J
KJ
D019149:Bioreactors; D018929:Cell Culture Techniques; D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D006801:Humans; D007371:Interferon-gamma / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins
D003470:Culture Media; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D007371:Interferon-gamma