Effect of preinduction specific growth rate on recombinant alpha consensus interferon synthesis in Escherichia coli.
The effect of preinduction specific growth rate on the yield of recombinant alpha consensus interferon in Escherichia coli K-12 was investigated. The cells used in the investigation contain a temperature-sensitive, walkaway plasmid bearing an insert that codes for alpha consensus interferon. Transcription of the recombinant gene is controlled by a lambda repressor/pL promoter system. The lambda promoter is regulated by the temperature-sensitive gene cI857 at 30 degrees C, but at 42 degrees C the promoter is derepressed. The cells were grown under glucose-limited conditions in a chemostat at pH 7 and 30 degrees C. Once steady state was achieved, the vessel temperature was raised to 42 degrees C and a fed-batch mode was initiated. Six dilution rates ranging from 0.025/h to 0.2/h were investigated. Cell dry weight, alpha consensus interferon content, glucose concentration, acetate concentration, and plasmid stability were measured. At each dilution rate, the expression level of alpha consensus interferon (g/g of cell dry wt) reached its maximum value approximately 3 h after induction. In addition, the expression level of alpha consensus interferon increases 4-fold as the dilution rate prior to induction is increased from 0.025/h to 0.2/h. Consequently, the expression of recombinant protein produced by E. coli is dependent on the preinduction specific growth rate.
Amgen, Inc., Thousand Oaks, California 91320.
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Curless
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Pope
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Tsai
D000085:Acetates / Q000378:metabolism; D003208:Concanavalin A; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005285:Fermentation; D005947:Glucose / Q000378:metabolism; D007370:Interferon Type I / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins; D013696:Temperature
D000085:Acetates; D007370:Interferon Type I; D011994:Recombinant Proteins; D003208:Concanavalin A; D005947:Glucose