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.
A two-stage, cyclic fed-batch fermentation process to produce recombinant human lymphokine was designed. The organism used in the study was Escherichia coli K-12 containing a temperature-sensitive walkaway plasmid bearing an insert which codes for a human lymphokine. Transcription of the recombinant gene is controlled by a lambda repressor/pL promoter system. The lambda promoter is regulated by the temperature-sensitive product of the cl857 gene at 30 degrees C, but at 42 degrees C the promoter is derepressed. The first or growth, stage of the process was maintained at 28 degrees C and operated in the fed-batch mode. The vessel was fed at a rate which gives a constant specific growth rate using a media designed to maintain a constant optical density OD(600) of 50. After the volume in the first stage reached the maximum working volume of the vessel (12 L), a portion of the vessel contents was transferred to the second stage. The second, or induction/product formation, stage also operated in the fed-batch mode, was kept at 42 degrees C, and was fed with a media that is conducive to recombinant human lymphokine synthesis. An optical density of more than 100 was consistently achieved in the second stage. Thirty cycles were completed with a consistent yield of human lymphokine and cell density in each cycle. The process was used to produce 200 L of OD(600) 50 material from the first stage in 10 days. The volumetric productivity (g lymphokine/L. day) of the two-stage, cyclic fed-batch process is twice that of a single-stage, fed-batch fermentation process.
Polyphosphate glass (sodium hexametaphosphate, sodium polyphosphate, glassy) was identified and tested as a source of phosphate that supports growth of recombinant Escherichia coli to cell densities over 110 g dry wt/L. Polyphosphate glasses cost no more than sodium and potassium phosphates, are more soluble than any other inorganic phosphate source, and do not form metallophosphate precipitates when mixed with other nutrients in proportions found in fermentation medias. Using a typical fermentation medium, 40% higher cell densities were obtained using polyphosphate glass rather than orthophosphate. Polyphosphate glasses will allow more flexibility in designing and executing high cell density fermentations.
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