期刊
CYTOTECHNOLOGY
卷 69, 期 2, 页码 371-389出版社
SPRINGER
DOI: 10.1007/s10616-017-0068-5
关键词
Antimicrobial protein; Galleria mellonella Gloverin; Stably transformed D; melanogaster S2 cells; Recombinant protein expression; Online process monitoring; Process optimization
资金
- Hessen State Ministry of Higher Education, Research and the Arts within Hessen initiative for scientific and economic excellence (LOEWE-Program) [LOEWE AZ: III L5- 518/19.004]
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 Gloverinfamily 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 mu M 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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