Rapid generation of Drosophila Schneider 2 (S2) cell lines and FACS-based isolation of high-yield soluble or membrane proteins.

Efficient production of milligram quantities of properly folded, functional eukaryotic proteins remains a significant challenge for structural and biochemical research, especially for membrane proteins and complex glycoproteins. The Drosophila Schneider 2 (S2) expression system provides a scalable, cost-effective alternative to baculovirus and mammalian platforms. However, its broader use is hampered by lengthy cell line development and variable expression levels. In this study, we present an optimized S2 expression workflow that reduces the time required to create stable cell lines from approximately 4 weeks to about 2 weeks without compromising protein yield. To further enhance expression, especially for low-expressing constructs, we used fluorescence-activated cell sorting (FACS) to select for high-expressing oligoclonal populations. Using a soluble human leucocyte antigen class II (HLA) and the transmembrane protein hCD81 as model proteins, FACS enrichment increased yields by ∼124-fold for the soluble protein and by ∼4.6-fold for the membrane protein. Protein functionality was confirmed through receptor-binding assays, SEC-MALS, and high-resolution crystallography for HLA, and ligand-binding assays for hCD81. Overall, this faster, scalable workflow enables rapid, high-yield production of structurally intact, functionally active soluble and membrane proteins from small culture volumes, establishing S2 cells as a fast and reliable platform for eukaryotic protein expression.