The Expression of Hemolytically Active Human Complement Protein C9 in Mammalian, Insect, and Yeast Cells

Topology of the membrane-bound form of complement protein C9 probed by glycosylation mapping, anti-peptide antibody binding, and disulfide modification

“…Hemolytic activity of complement proteins was assayed according to standard procedures and bactericidal activity using the Escherichia coli strain C600 was assayed as described (Tomlinson et al, 1993). Cell survival is presented as the ratio of CFU/mL incubated with serum (sample) to CFU/mL of cells incubated in buffer (control).…”

“…The previously characterized C9 vectors pSL301HuC9 (Tomlinson et al, 1993) and pYW29 (Wang et al, 2000), which contains two mutations (L532S/K538R) and a His6 C-terminal tag, were used as the starting plasmids for the expression vectors listed in Figure 1. A KpnI – SacI fragment from pSL301HuC9 was cloned into pSelect-1 to eliminate the two existing N-glycosylation sites individually using the “Altered Sites Mutagenesis System” (Promega, Madison, WI).…”

“…Transposition of the various C9 genes was carried out by transforming the recombinant donor plasmids into E. coli DH10B harboring the transposition helper plasmid pMon7124 and the bacmid bMON14272. Selection of the correct composite bacmids was performed as described (Tomlinson et al, 1993) and purified bacmid DNA was used for the transfection of insect cell lines.…”

“…COS-7 cells were transfected with the pSVL-derived expressions vectors by electroporation and the different proteins were isolated as described previously (Tomlinson et al, 1993). C9 mutants were expressed at 27 °C by baculovirus-infected Sf9 cells growing in suspension in serum-free medium contained in 250 mL shaker flasks rotating at 135 rpm as described (Rossi et al, 1998).…”

“…C9 mutants were expressed at 27 °C by baculovirus-infected Sf9 cells growing in suspension in serum-free medium contained in 250 mL shaker flasks rotating at 135 rpm as described (Rossi et al, 1998). Protein purifications were performed as published (Tomlinson et al, 1993) except those that carried a C-terminal hexahistidine tag were purified by metal-affinity chromatography using the manufacturer's (Qiagen, Valencia, CA) protocol.…”