Abstract
Human granulocyte colony-stimulating factor (hG-CSF) is a cytokine that regulates the proliferation, maturation, and differentiation of precursor cells to neutrophils. In the present study, we report the feasibility of inducing recombinant hG-CSF expression (rhG-CSF) in a pET vector system by combinatorial induction using low-concentration ethanol, IPTG, and lactose and auto-induction media (AIM). The coding sequence of hG-CSF transcript variant 2 was expressed in pET14 vector, and the effect of combinatorial induction was analyzed on inclusion body (IB) formation, biomass, protein purification, and bioactivity. Results showed that there was an inverse relationship between the temperature and soluble expression of rhG-CSF. Three-step washing with Triton-X, 2 M, and 5 M urea resulted in the maximum recovery of IBs. Combinatorial single-spike induction with IPTG, ethanol, and lactose in a batch culture led to a 3-fold increase in the expression of rhG-CSF. It was also observed that low concentration of ethanol (1–3% v/v) could be used in lieu of IPTG for inducing the rhG-CSF protein expression without adversely affecting biomass production. A 2.4-fold increase in productivity was obtained in LB-AIM media with combinatorial ethanol induction, and the overall yield of 2.8 g/L rhG-CSF was found. The purified rhG-CSF was bioactive and increased the cellular proliferation of umbilical cord blood-derived mesenchymal stem cells (U-MSC) by 29%. In conclusion, our study shows that combined ethanol induction can enhance the expression of rhG-CSF with three-step washing for recovery of the proteins from IBs and a single-step purification of rhG-CSF by affinity chromatography.
Key points
• Low concentration of ethanol (1–3%) could be used in lieu of IPTG for inducing rhG-CSF expression.
• Combinatorial single-spike induction with IPTG, ethanol, and lactose improved rhG-CSF expression.
• Purified rhG-CSF was bioactive and increased the proliferation of U-MSC.
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This work was funded by Department of Biotechnology (#BT/PR9535/AAQ/1/569/2013), Government of India.
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BM, GM, and BS performed the experiments; BM, GM, BS, and MKG analyzed the results; SJU and MKG conceptualized the work; BM and MKG wrote the manuscript; SJU and MKG reviewed the work. BM, GM, BS, SJU, and MKG reviewed and finalized the manuscript for submission. MKG provided resources and overall supervised the work.
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Mishra, B., Murthy, G., Sahoo, B. et al. Combinatorial ethanol treatment increases the overall productivity of recombinant hG-CSF in E. coli: a comparative study. Appl Microbiol Biotechnol 104, 9135–9145 (2020). https://doi.org/10.1007/s00253-020-10899-z
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DOI: https://doi.org/10.1007/s00253-020-10899-z