Expression and characterization of recombinant bovine C3d fusion proteins
The gene fragment encoding bovine complement component C3d (boC3d) was isolated, cloned, sequenced and expressed in preparation for investigation of its potential as an adjuvant in cattle. Studies in mice attribute the adjuvant activity of C3d to a lowered B-cell activation threshold, resulting from the cross-linking of membrane-Ig and the B-cell co-receptor complex CD21(CR2)/CD19/CD81. To evaluate binding of recombinant boC3d to native bovine CD21, a recombinant boC3d protein was generated that included a biotinylation signal sequence at the N-terminal end of the boC3d sequence, for endogenous biotinylation by 'E. coli' via the BirA holoenzyme synthetase. Three additional constructs containing a portion of the gene encoding the leukotoxin produced by 'Mannheimia haemolytica' A1 linked to one, two or three boC3d units were created for expression in 'E. coli' as recombinant fusion proteins, as potential vaccines. All recombinant proteins incorporated polyhistidine tags and were purified by Ni-NTA agarose chromatography, then identified by SDS-PAGE and western blot. These proteins will provide the basis for future studies of receptor binding and cell-activation, and for immunization trials in cattle.