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Cytotechnology

, Volume 69, Issue 2, pp 371–389 | Cite as

Optimized expression of the antimicrobial protein Gloverin from Galleria mellonella using stably transformed Drosophila melanogaster S2 cells

  • Jan Zitzmann
  • Tobias Weidner
  • Peter Czermak
Original Article

Abstract

Antimicrobial proteins and peptides (AMPs) are valuable as leads in the pharmaceutical industry for the development of novel anti-infective drugs. Here we describe the efficient heterologous expression and basic characterization of a Gloverin-family AMP derived from the greater wax moth Galleria mellonella. Highly productive single-cell clones prepared by limiting dilution achieved a 100% increase in productivity compared to the original polyclonal Drosophila melanogaster S2 cell line. Comprehensive screening for suitable expression conditions using statistical experimental designs revealed that optimal induction was achieved using 600 µM CuSO4 at the mid-exponential growth phase. Under these conditions, 25 mg/L of the AMP was expressed at the 1-L bioreactor scale, with optimal induction and harvest times ensured by dielectric spectroscopy and the online measurement of optical density. Gloverin was purified from the supernatant by immobilized metal ion affinity chromatography followed by dialysis. In growth assays, the purified protein showed specific antimicrobial activity against two different strains of Escherichia coli.

Keywords

Antimicrobial protein Galleria mellonella Gloverin Stably transformed D. melanogaster S2 cells Recombinant protein expression Online process monitoring Process optimization 

Notes

Acknowledgements

We would like to thank the Hessen State Ministry of Higher Education, Research and the Arts (Grant No. LOEWE AZ: III L5-518/19.004) for financial support within the Hessen initiative for scientific and economic excellence (LOEWE-Program). The authors acknowledge Dr. Richard M. Twyman for editing the paper.

Author contributions

JZ conceived, designed and conducted the experiments, and wrote the manuscript; TW helped to draft and to revise the manuscript; PC helped to draft and to revise the manuscript, and supervised the research; All authors have given their approval for this final version of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analysis, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

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© Springer Science+Business Media Dordrecht 2017

Authors and Affiliations

  1. 1.Institute of Bioprocess Engineering and Pharmaceutical TechnologyUniversity of Applied Sciences MittelhessenGiessenGermany
  2. 2.Department of Chemical EngineeringKansas State UniversityManhattanUSA
  3. 3.Faculty of Biology and ChemistryJustus-Liebig University of GiessenGiessenGermany
  4. 4.Project Group BioresourcesFraunhofer Institute for Molecular Biology and Applied Ecology (IME)GiessenGermany

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