1. Law S. K., Dodds A. W., Protein Sci.6, 263 (1997).
2. M. B. Fischer, et al., J. Immunol. 157, 549 (1996); Ahearn J. M., et al., Immunity 4, 251 (1996).
3. Fearon D. T., Carter R. H., Annu. Rev. Immunol.13, 127 (1995).
4. C3d of Complement as a Molecular Adjuvant: Bridging Innate and Acquired Immunity
5. Expression and purification were done as follows. A human C3d cDNA corresponding to residues 996 through 1303 containing the Cys 1010 → A 1010 mutation (14) was inserted into the bacterial expression plasmid pET 15b (Novogen) in which the NH 2 -terminal oligo-His encoding sequence has been deleted. The encoded C3d fragment contained an additional eight amino acids (MLDAERLK) (21) at the NH 2 -terminus that would not be present in enzymatically produced C3d ML being vector derived and DAERLK corresponding to residues at the COOH-terminus of C3g. Protein expression in transformed E. coli strain BL21 (DE3) grown in Luria Bertoni broth with ampicillin was induced with 0.25 mM isopropyl-β- d -thiogalactopyranoside (IPTG) at 28°C for 12 hours. Selenomethionine (SeMet) C3d was expressed in the same cells grown in ampicillin-containing M9 minimal medium supplemented with 0.4% glucose MgSO 4 (100 mg/ml) CaCl 2 (15 mg/ml) and thiamine (1.25 mg/ml). After IPTG induction the medium was further supplemented with the amino acids K L I V T and F (100 mg/ml each) and seleno- l -methionine (50 mg/ml) (22). Both native C3d and the SeMet C3d were purified from the soluble fraction of the bacterial lysate by DEAE-Sephacel then by Mono Q HR10/10 fast protein liquid chromatography (FPLC) (Pharmacia) both at pH 7.1 and finally by Mono S HR5/5 FPLC (Pharmacia) at pH 6. Amino acid composition analysis of SeMet C3d showed that the replacement of the eight Met positions by SeMet was near quantitative. Recombinant C3d Cys 1010 → Ala 1010 was assessed for binding to human Raji B cell–associated CR2 with a rosette inhibition assay (17). Serum-derived C3dg was used as a positive control. As monomeric ligands both proteins inhibited 50% of rosette formation at 0.2 μM.