title: Optimized expression of the antimicrobial protein Gloverin from Galleria mellonella using stably transformed Drosophila melanogaster S2 cells.
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 CuSO4at 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.
authors.affiliation: Institute of Bioprocess Engineering and Pharmaceutical Technology, University of Applied Sciences Mittelhessen, Giessen, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Zitzmann
authors.affiliation: Institute of Bioprocess Engineering and Pharmaceutical Technology, University of Applied Sciences Mittelhessen, Giessen, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Weidner
authors.affiliation: Institute of Bioprocess Engineering and Pharmaceutical Technology, University of Applied Sciences Mittelhessen, Giessen, Germany. peter.czermak@lse.thm.de.|Department of Chemical Engineering, Kansas State University, Manhattan, KS, USA. peter.czermak@lse.thm.de.|Faculty of Biology and Chemistry, Justus-Liebig University of Giessen, Giessen, Germany. peter.czermak@lse.thm.de.|Project Group Bioresources, Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Giessen, Germany. peter.czermak@lse.thm.de.
authors.forename: Peter
authors.identifier: http://orcid.org/0000-0001-7485-500X
authors.initials: P
authors.lastname: Czermak
keywords: Antimicrobial protein; Galleria mellonella Gloverin; Online process monitoring; Process optimization; Recombinant protein expression; Stably transformed D. melanogaster S2 cells