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

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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.

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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.

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Authors and Affiliations

  1. Institute of Bioprocess Engineering and Pharmaceutical Technology, University of Applied Sciences Mittelhessen, Giessen, Germany

    Jan Zitzmann, Tobias Weidner & Peter Czermak

  2. Department of Chemical Engineering, Kansas State University, Manhattan, KS, USA

    Peter Czermak

  3. Faculty of Biology and Chemistry, Justus-Liebig University of Giessen, Giessen, Germany

    Peter Czermak

  4. Project Group Bioresources, Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Giessen, Germany

    Peter Czermak

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  1. Jan Zitzmann
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  3. Peter Czermak
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Correspondence to Peter Czermak.

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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.

Appendices

Appendix 1: Statistical assessment of normality and homoscedasticity for the comparison of the expression of the parental polyclonal cells and the monoclonal cell line D7

Statistical assessment of expression data was conducted using R v3.2.3 (R Core Team 2014). Because the normal q–q plots in Fig. 10 do not show any points outside their confidence intervals, there is no reason to doubt the validity of the assumption of normality for the data in both groups. The high p value of 0.9619 in a subsequent F-test indicates that there is insufficient evidence to reject the assumption of homoscedasticity. Consequently, Student’s two-sample t test was used to compare the means of both datasets.

Fig. 10
figure 10

Normal q–q plots for the expression datasets used to compare the monoclonal cell line and polyclonal culture

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Appendix 2: Response surface method for the determination of optimal induction conditions

See Tables 1 and 2.

Table 1 ANOVA for the quadratic model that describes GmGlv production after 4 days as a function of cell concentration (A) and CuSO4 concentration (B)
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Table 2 ANOVA for the cubic model that describes cell viability after 4 days as a function of cell concentration (A) and CuSO4 concentration (B)
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Appendix 3: Time course of additional process variables during the cultivation of rS2 cells for the production of GmGlv

See Fig. 11.

Fig. 11
figure 11

Time course of standard bioreactor parameters: a stirrer speed (n, black), dissolved oxygen (pO2, red), gas mix composition (blue) and pH (gray). b Glucose consumption during cultivation

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Appendix 4: Comparison of the induction conditions in 37 studies

See Table 3.

Table 3 Comparison of the induction conditions in 37 studies using the Mt promoter in rS2 cells
Full size table

Appendix 5: Site-specific binding of GmGlv to LPS

There is no broad agreement on the exact position at which Gloverin binds to LPS. The latter comprises three major parts: lipid A, a carbohydrate core region, and the O-specific antigen. If all parts are present, the LPS is regarded as smooth. If the outer parts are absent, LPS is regarded as rough or deep rough depending on the degree of truncation (Ra, Rc, Rd, and Re LPS). BmGv is inactive against E. coli DH5alpha but active against the rough mutants E. coli D21 (Ra-LPS), E. coli D21e7 (Rc-LPS), E. coli D21f1 (Rd-LPS) and E. coli D21f2 (Re-LPS) (Yi et al. 2013). Furthermore, TnGlv (Lundström et al. 2002) and HgGlv (Axén et al. 1997) also show activity primarily against E. coli D21f2. These results indicate that most (positively charged) Gloverins probably bind to the (negatively charged) inner core region or lipid A. The two E. coli strains used in this study have not been comprehensively characterized in terms of their LPS phenotype, but their parental stains carry short LPS (Chart et al. 2000). Furthermore E. coli Rosetta Gami 2 carries the galE–galK mutation, which blocks the synthesis of UDP-galactose and its incorporation in the LPS, thus producing a truncated LPS (van Die et al. 1984). These data suggest that GmGlv also binds to the inner part of the LPS. However, MsGlv binds to the outer core region and the O-specific antigen of LPS and also to other fungal and bacterial membrane compounds, but it does not interact with Rc, Rd or Re LPS or with lipid A (Xu et al. 2012).

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Zitzmann, J., Weidner, T. & Czermak, P. Optimized expression of the antimicrobial protein Gloverin from Galleria mellonella using stably transformed Drosophila melanogaster S2 cells. Cytotechnology 69, 371–389 (2017). https://doi.org/10.1007/s10616-017-0068-5

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