Overview Introduction The Drosophila Expression System (DESÆ) utilizes a cell line derived from Drosophila melanogaster, Schneider 2 (S2) cells, and a simple plasmid vector for the expression of heterologous proteins. S2 cells are easily maintained in loosely adherent or suspension culture at room temperature and do not require CO2. The vectors used for expression in S2 cells are very versatile, allowing inducible expression from the metallothionein (MT) promoter (Bunch et al., 1988; Maroni et al., 1986) or constitutive expression from the (actin) Ac5 promoter (Chung and Keller, 1990). Expression can either be intracellular or secreted for simplified purification. Many native signal sequences are functional in S2 cells and can be used to secrete proteins using either the pAc5.1/V5-His or pMT/V5-His vectors. pMT/BiP/V5-His is also available if you want to fuse your protein to a Drosophila secretion signal sequence. To facilitate cloning of Taq-amplified PCR products, the pMT/V5-His vector is available adapted to topoisomerase I. In addition, each expression vector encodes a C-terminal peptide containing the V5 epitope for antibody detection and a polyhistidine (6xHis) tag for purification. Stable cell lines expressing heterologous proteins can be generated in 3-4 weeks from a single cotransfection of the expression vector and the pCoHygro selection vector or 2 weeks following cotransfection of the expression vector and the pCoBlast selection vector. By optimizing the ratio of expression vector to selection vector, cell lines with a very high copy number of the desired gene can be generated, leading to increased expression levels of the desired protein.