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Single-cell cloning enables the selection of more productive Drosophila melanogaster S2 cells for recombinant protein expression

: Zitzmann, J.; Schreiber, C.; Eichmann, J.; Bilz, R.O.; Salzig, D.; Weidner, T.; Czermak, P.

Volltext ()

Biotechnology Reports 19 (2018), Art.e00272, 10 S.
ISSN: 2215-017X
Zeitschriftenaufsatz, Elektronische Publikation
Fraunhofer IME ()

The generation of monoclonal cell lines is an important early process development step for recombinant protein production. Although single-cell cloning is an established method in mammalian cell lines, straightforward protocols are not yet available for insect cells. We describe a new method for the generation of monoclonal insect cells without using fetal bovine serum and/or feeder cells pretreated by irradiation or exposure to mitomycin. Highly productive clones of Drosophila melanogaster S2 cells were prepared in a two-step procedure, comprising the establishment of a polyclonal population and subsequent single cell isolation by limiting dilution. Necessary growth factors were provided by co-cultivation of single transformants with untransfected feeder cells, which were later removed by antibiotic selection. Enhanced expression of EGFP and two target peptides was confirmed by flow cytometry and dot/western blotting. Highly productive clones were stable, showed a uniform expression profile and typically a sixfold to tenfold increase in cell-specific productivity.