Figure 2

Expression of recombinant gp41 in human cells and analysis of the released proteins. (A-C) Immunofluorescence analysis of 293 cells transfected with (A) backbone vector pcDNA3.1(−) as negative control, (B) expression vector coding for wt gp41, (C) and gp41 with the mutation Q2A in the isu domain. Proteins were detected using the mab 2F5. (D) Comparison of the wt gp41 and the mutant gp41(L1A) with gp41ΔCT (lacking the cytoplasmatic tail) by SDS-PAGE/Western blot analysis. Proteins in the supernatant were concentrated 20 times using 30 kDa cut-off membranes. (E) Evidence for glycosylation of the gp41 and gp41ΔCT produced in 293 cells. Proteins were treated with PNGase F (T), Nt – not treated, c – cells transfected with control vector. p35 is the non-glycosylated form of gp41 and p17 is the non-glycosylated form of gp41ΔCT. (F) Evidence for trimerisation of the wt gp41, selected mutants and the gp41ΔCT. After transfection of 293 T cells the proteins were concentrated 20 times as described above and a 4%-20% gradient native PAGE/Western blot analysis was performed. Positions of the trimers (arrow heads) and the molecular markers are given (bars). (G) Expression of wt gp41 and gp41 with different mutations in the isu domain in transfected 293 T cells grown in FCS-free medium (upper panel) and proteins recovered from FCS-free medium 48 hours post transfection and concentrated 20 times as described (lower panel). For technical reasons, E9A was analyzed in an additional experiment. A 4-20% gradient SDS-PAGE/Western blot analysis was performed with mab 2F5. Arrow heads indicate the position of gp41. “c” in panels D, E, F and G indicate concentrated supernatant from cells transfected with the backbone vector.