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Optimization of the AT-content of Codons Immediately Downstream of the Initiation Codon and Evaluation of Culture Conditions for High-level Expression of Recombinant Human G-CSF in Escherichia coli

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Abstract

Enhanced therapeutic importance of recombinant human granulocyte colony stimulating factor (rhG-CSF) has encouraged us to develop a processing method for its high-level expression in E. coli. In this study, we established a high-yielding clone by incorporation of silent mutations at N-terminal region of human G-CSF gene. We studied and optimized various parameters of culture conditions connected with the expression of rhG-CSF. The maximum expression was obtained in a defined medium supplemented with 1% glucose. The gene in pET-3a vector in E. coli BL21 (DE3) PLysS host strain was induced with 2 mM isopropyl β-d-1-thiogalacto pyronoside. The cell growth and productivity was enhanced about 1.6- and 1.5-folds, respectively when inducing the culture at OD600 value of 6 than 2. The protein expression was significantly increased by addition of rifampicin at concentration of 200 μg/ml. The AT content of 51.8% with suitable codon sequences at N-terminal region and the concentration of rifampicin were identified as the key factors with a significant impact on protein expression. The specific productivity of 104 mg/OD/l (68.7% of total cellular protein) of rhG-CSF was obtained toward the end of the study, which is almost 1.5 times higher yield than reported so far in the literature.

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Acknowledgements

The authors are highly thankful to the authorities of Natco Pharma Limited, Hyderabad for providing support in all aspects of this work. The technical support from the Analytical Development Division of Natco Research Centre is gratefully acknowledged.

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Authors and Affiliations

  1. Research and Development, Biotechnology Division, Natco Research Centre, Hyderabad, 500018, India

    Dasari V. Krishna Rao & Adibhatla Kali S. Bhujanga Rao

  2. Institute of Science and Technology, Department of Biotechnology, Jawaharlal Nehru Technological University, Hyderabad, 500072, India

    Joginapally V. Rao & Mangamoori L. Narasu

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  1. Dasari V. Krishna Rao
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Krishna Rao, D.V., Rao, J.V., Narasu, M.L. et al. Optimization of the AT-content of Codons Immediately Downstream of the Initiation Codon and Evaluation of Culture Conditions for High-level Expression of Recombinant Human G-CSF in Escherichia coli . Mol Biotechnol 38, 221–232 (2008). https://doi.org/10.1007/s12033-007-9018-3

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