Rabies virus glycoprotein expression in Drosophila S2 cells. I. Functional recombinant protein in stable co-transfected cell line

Recombinant rabies virus glycoprotein (rRVGP) was expressed in Drosophila melanogaster Schneider 2 (S2) cells. The cDNA encoding the entire RVGP gene was cloned in an expression plasmid under the control of the constitutive actin promoter (Ac), which was co-transfected into S2 cells together with a hygromycin selection plasmid. Selected S2 cell populations (S2AcRVGP) had a decreased ability to grow and consume substrates, when compared to the non-transfected cells (S2). They were shown, by PCR, to express the RVGP gene and mRNA and, by immunoblotting, to synthesize the rRVGP in its expected molecular mass of 65 kDa. ELISA kinetic studies showed the rRVGP expression in cell lysates and supernatants attaining concentrations of 300 microg/L. By flow cytometry analysis, about 30% of the cells in the co-transfected populations were shown to express the rRVGP. Cell populations selected by limiting dilution expressed higher rRVGP yields. Mice immunized with rRVGP were shown to synthesize antibodies against rabies virus and be protected against experimental infection with rabies virus. The data presented here show that S2 cells can be suitable hosts for the rRVGP expression, allowing its synthesis in a high degree of physical and biological integrity.     

Analytical approach for the extraction of recombinant membrane viral glycoprotein from stably transfected Drosophila melanogaster cells

Parameters for storage, lysis and concentration of Drosophila melanogaster Schneider 2 (S2AcRVGP) cells expressing the recombinant rabies virus glycoprotein (RVGP) were studied with regard to RVGP quantification by ELISA, for productivity evaluation and future purification. Lysis buffers were formulated with Tris, NaCl, glycerol, EDTA, KCl, Na(2)PO(4), MgCl(2), PMSF and NP-40 or CHAPS. S2AcRVGP cells (10(7) cells at the exponential growth phase) were frozen at -20 degrees C as a dry pellet, suspended in buffer (B) formulations or after treatment with lysis buffer (LB) formulations. They were then thawed as cell pellets or with B formulations or PBS at 4 degrees C or at room temperature and then lysed with LB formulations. For RVGP quantification by ELISA, a protocol was chosen of cell preparation including cell freezing as dry pellet, cell thawing at 4 degrees C with B4 (Tris, NaCl, MgCl(2), PMSF) and cell lysis with the LB4 (B4 + NP-40) since it fulfilled requirements of high RVGP detection, and was easily performed with mixtures freezing quickly, and a cost-saving LB formulation could be used. Using these established conditions, we examined the optimal cell concentration for RVGP quantification by ELISA. Results showed that an increase in the RVGP detection (from 62.5 to 1083 ng/10(7) cells) paralleled a decrease in the cell number (3 x 10(7) - 10(5) cells) used. The NP-40 concentration present in the LB4 was further investigated as a function of the cell number used for sample preparation. Previous results were confirmed indicating that higher NP-40 concentrations led to a decreased detection of RVGP. Altogether our data clearly pointed out the crucial effects of cell freeze, thaw, lysis and concentration on immune detection of recombinant membrane glycoproteins and can be useful as a guideline for sample preparation for this purpose.     

Identifying calpain substrates in intact S2 cells of Drosophila

Calpains are cysteine proteases involved in a number of physiological and pathological processes, yet our knowledge of substrates cleaved in vivo, in intact cells, is scarce. In this work we made an attempt to develop a technique for finding calpain substrates in intact Drosophila Schneider S2 cells. The procedure consists in comparative 2D gelelectrophoresis: three identical samples were treated in different ways: A (control, no addition), B, activated (Ca²⁺ and ionomycin added), C, inactivated (additions as in B + specific calpain inhibitor). 2D gel pattern were analyzed by densitometry. Spots showing density relation A > B << C were identified by mass spectroscopy. In a typical run, 11 candidate substrates were recognized; out of these, four were randomly selected: all four were verified to be calpain substrates, by digestion of the recombinant protein with recombinant calpain.     

Expression and immunogenicity of recombinant glycoprotein D of herpes simplex virus 1 in Drosophila S2 cells

Herpes simplex virus type 1 (HSV-1) is responsible for cold sores in the general population, but also contributes to the development of other more serious diseases in some circumstances. The viral glycoprotein D (gD) is essential for virus entry into host cells. In the present study, the Drosophila melanogaster Schneider 2 (S2) expression system (DES) was evaluated for the expression of recombinant gD1. The DNA sequences encoding the full-length gD1 (369aa, FLgD1) and a truncated gD1 form corresponding to the ectodomain (314aa, EgD1) were cloned into S2 expression vector pMT/BiP/V5-HisA to generate pMT-EgD1 and pMT-FLgD1, respectively. Two forms of gD1 gene were fitted with a hexahistidine tag to facilitate their purification. Cell populations expressing the highest gD1 levels were selected by using a limiting dilution assay. Western blot, flow cytometry (FACS), and confocal immunofluoresence assay demonstrated that the full-length form is restrained in the lipid membranes of the cell and the ectodomain form is secreted into the medium. Recombinant ectodomain gD1 was scaled up and purified from the culture medium using nickel nitrilotriacetic acid affinity chromatography, and a maximum production level of 56.8 mg/L of recombinant gD1 was obtained in a shake-flask culture of S2 cells after induction with 5 µM CdCl2 for 4 days. Mice were then immunized with recombinant purified gD1 and produced high titers of antibody measured by enzyme-linked immunosorbent assay (ELISA; 1:5,120,000) as well as high plaque neutralization titer (1:320). Overall, the data indicated that stable expression in S2 cells is a practical way of synthesizing gD1 for use in structural and functional studies in the further study.     

Temperature and developmental responses of body and cell size in Drosophila; effects of polyploidy and genome configuration

Increased adult body size in Drosophila raised at lower temperatures could be attributed both to an increase in the cell volume and cell number. It is not clear, however, whether increased cell size is related to (or even caused by) increased nuclear volume and genome size (or configuration). Experiments with Drosophila melanogaster stocks (Oregon-R and w1118) raised at 16, 22, 24, and 28 °C resulted in larger adult body and wing size with lower temperature, while eye size was less affected. The increase in wing size reflected an increase in cell size in both males and females of both stocks. The nucleus size, genome size, and DNA condensation of adult flies, embryos, and Schneider 2 cells (S2 cells, of larval origin) were estimated by flow cytometry. In both adult flies and S2 cells, both nucleus size and DNA condensation varied with temperature, while DNA content appears to be constant. From 12% to 18% of the somatic cells were tetraploid (4C) and 2–5% were octoploid (8C), and for the Oregon strain we observed an increase in the fraction of polyploid cells with decreasing temperature. The observed increase in body size (and wing size) at low temperatures could partly be linked with the cell size and DNA condensation, while corresponding changes in the haploid genome size were not observed.