Optimized expression of the antimicrobial protein Gloverin from Galleria mellonella using stably transformed Drosophila melanogaster S2 cells
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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.
KeywordsAntimicrobial protein Galleria mellonella Gloverin Stably transformed D. melanogaster S2 cells Recombinant protein expression Online process monitoring Process optimization
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.
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.
- Cherbas L, Cherbas P (2007) Transformation of Drosophila cell lines. In: Murhammer D (ed) Baculovirus and insect cell expression protocols. Humana Press, New York, pp 317–340Google Scholar
- Chiou M-J, Chen L-K, Peng K-C et al (2009) Stable expression in a Chinese hamster ovary (CHO) cell line of bioactive recombinant chelonianin, which plays an important role in protecting fish against pathogenic infection. Dev Comp Immunol 33:117–126. doi: 10.1016/j.dci.2008.07.012 CrossRefGoogle Scholar
- Dorner AJ, Wasley LC, Kaufman RJ (1989) Increased synthesis of secreted proteins induces expression of glucose-regulated proteins in butyrate-treated Chinese hamster ovary cells. J Biol Chem 264:20602–20607Google Scholar
- Druzinec D, Weiss K, Elseberg C et al (2014) Process analytical technology (PAT) in insect and mammalian cell culture processes: dielectric spectroscopy and focused beam reflectance measurement (FBRM). In: Pörtner R (ed) Animal cell biotechnology. Humana Press, New York, pp 313–341CrossRefGoogle Scholar
- Graziano MP, Broderick DJ, Tota MR (1998) Expression of G protein-coupled receptors in Drosophila Schneider 2 cells. In: Lynch KR (ed) Receptor biochemistry and methodology series, identification and expression of G protein-coupled receptors. Wiley, New York, pp 181–195Google Scholar
- Lemos MAN, dos Santos AS, Astray RM et al (2009) Rabies virus glycoprotein expression in Drosophila S2 cells. I: design of expression/selection vectors, subpopulations selection and influence of sodium butyrate and culture medium on protein expression. J Biotechnol 143:103–110. doi: 10.1016/j.jbiotec.2009.07.003 CrossRefGoogle Scholar
- Park JH, Kyung-Hwa C, Lee YH, Hae-Yeong K, Jai-Myung Y, In-Sik C (2002) Production of recombinant rotavirus capsid protein VP7 from stably transformed Drosophila melanogaster S2 cells. J Microbiol Biotechnol 12:563–568Google Scholar
- Perret BG, Wagner R, Lecat S, Brillet K, Rabut G, Bucher B et al (2003) Expression of EGFP-amino-tagged human mu opioid receptor in Drosophila Schneider 2 cells: a potential expression system for large-scale production of G-protein coupled receptors. Protein Expr Purif 31:123–132CrossRefGoogle Scholar
- R Core Team (2014) R: a language and environment for statistical computing. R Foundation for Statistical Computing, ViennaGoogle Scholar
- Schetz JA, Shankar EPN (2004) Protein expression in the Drosophila Schneider 2 cell system. In: Gerfen CR, Holmes A, Sibley D, Skolnick P, Wray S (eds) Current protocols in neuroscience. Wiley, New York, pp 4.16.1–4.16.15Google Scholar
- Schneider I (1972) Cell lines derived from late embryonic stages of Drosophila melanogaster. J Embryol Exp Morphol 27:353–365Google Scholar
- Seok YJ, Kim KI, Yoo KH, Hwang-Bo J, Lee HH, Shon DH et al (2010) Expression and immunogenicity of a recombinant chimeric protein of human colorectal cancer antigen GA733-2 and an Fc antibody fragment in stably transformed Drosophila melanogaster S2 cells. Appl Biochem Biotechnol 162:1435–1445CrossRefGoogle Scholar
- van Die IM, Zuidweg EM, Bergmans JE, Hoekstra WP (1984) Transformability of galE variants derived from uropathogenic Escherichia coli strains. J Bacteriol 158:760–761Google Scholar