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Supplementation of Substrate Uptake Gene Enhances the Expression of rhIFN-β in High Cell Density Fed-Batch Cultures of Escherichia coli

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Abstract

Over-expression of recombinant proteins in Escherichia coli triggers a metabolic stress response which causes a sharp decline in both growth and product formation rates post induction. We identified a key down-regulated substrate utilization gene, glycerol kinase (glpK), whose up-regulation could help alleviate this stress response. In a proof of principal study conducted in shake flask cultures, the glpK gene under the “ara” promoter in a pPROLar.A122 vector was co-transformed along with the recombinant interferon-β (rhIFN-β) gene in a pET22b vector into E. coli BL-21(DE3) cells. Co-expression of glpK improved the expression levels of rhIFN-β in glycerol containing medium, while no such gain was observed in medium without glycerol. This study was extended to high cell density fed-batch cultures where exponential feeding of complex substrates was done to increase biomass and hence product titers. For this we first constructed a modified E. coli strain BL-21(glpK +) where the glpK gene was inserted downstream of the ibpA promoter in the host chromosome. There was a significant improvement in growth as well as expression levels of rhIFN-β in this modified strain when the feed medium contained high glycerol. A final product concentration of 4.8 g/l of rhIFN-β was obtained with the modified strain which was 35 % higher than the control.

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Acknowledgments

Financial support by Department of Biotechnology, Government of India is gratefully acknowledged. We would also like to kindly acknowledge Prof Hermann Bujard, (ZMBH) Universitaet Heidelberg for the plasmid pPROLar.A122 and Dr. Sara L. Vassallo, Harvard Medical School, Boston for the plasmid pKOV.

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  1. School of Biotechnology, Jawaharlal Nehru University, New Delhi, 110067, India

    Anuradha B. Singh & Krishna J. Mukherjee

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  1. Anuradha B. Singh
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  2. Krishna J. Mukherjee
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Correspondence to Krishna J. Mukherjee.

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Singh, A.B., Mukherjee, K.J. Supplementation of Substrate Uptake Gene Enhances the Expression of rhIFN-β in High Cell Density Fed-Batch Cultures of Escherichia coli . Mol Biotechnol 54, 692–702 (2013). https://doi.org/10.1007/s12033-012-9611-y

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