Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
9902241 | Journal of Immunological Methods | 2005 | 16 Pages |
Abstract
Proteins fused to activated complement (C) fragments elicit enhanced immunogenicity. This “natural adjuvant” effect may have important implications when considering novel vaccination approaches. Here we describe both the construction of a novel fusion protein, consisting of a well characterized test antigen fused to multiple copies of the activated complement component (C3d)3, as well as an efficient method for its expression and production in insect cells. Using the inherent biological advantages of the baculovirus expression system, as well as applying specific infection and harvesting modifications, we have optimized the efficiency of protein production. Our modifications allow purification of fusion proteins directly from cell supernatant in a single anion exchange chromatographic step. This alleviates the requirement for the inclusion of protein affinity tags. The integrity of the purified recombinant protein was evaluated by SDS PAGE analysis, reactivity with antibodies, as well as in vivo by administration as an immunogen.
Keywords
PBSBEVSCR2FCSFDCBCRenzyme linked immunosorbant assayAdjuvantImmunogenicityimmunoglobulin Protein expressionELISAfoetal calf serumB cellsFollicular dendritic cellsTetanus toxinbaculovirus expression vector systemPhosphate buffered salineComplementmajor histocompatibility complexMHCAntigenB cell antigen receptorcomplement receptor 2
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Authors
Denise V. Barrault, Michael Steward, Vivienne F. Cox, Richard A.G. Smith, Andrew M. Knight,