51. Expression in insect cells of the functional domain of CD21 (complement receptor type two) as a truncated soluble molecule using a baculovirus vector.
- Author
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Prodinger WM, Schoch J, Schwendinger MG, Hellwage J, Parson W, Zipfel PF, and Dierich MP
- Subjects
- Animals, Antibodies, Monoclonal genetics, Antibodies, Monoclonal immunology, Antigen-Antibody Reactions, B-Lymphocytes immunology, B-Lymphocytes metabolism, Female, Genetic Vectors immunology, Humans, Immunization, Mice, Mice, Inbred BALB C, Plasmids immunology, Receptors, Complement immunology, Receptors, Complement 3d immunology, Recombinant Proteins genetics, Recombinant Proteins immunology, Spodoptera, Baculoviridae genetics, Genetic Vectors genetics, Plasmids genetics, Receptors, Complement genetics, Receptors, Complement 3d biosynthesis
- Abstract
We have made use of the plasmid vector pBSV-8His recently established for baculovirus-mediated expression of His-tagged proteins for the production of a truncated soluble complement regulator protein. The protein comprised the N-terminal part, i.e. short consensus repeats (SCRs) 1-4, of the B-cell membrane protein complement receptor type two (CR2; CD21) and contained the functional epitopes which mediate the binding of the complement component C3 fragments C3dg and iC3b. This recombinant protein, termed rsCR2.1-4, was furnished with a C-terminal histidine-tag for easy purification from insect cell supernatant. The yield of > 90% pure rsCR2.1-4 was 3 micrograms/ml supernatant at day eight p.i. RsCR2.1-4 was expressed as two proteins with a M(r) of 29 and 31 representing differentially glycosylated forms. Both reacted specifically with anti-CR2 mAb HB5 directed against SCRs 3-4, but not with anti-CR2 mAbs recognizing SCRs beyond SCR 4. RsCR2.1-4 was able to bind C3dg and to block binding of C3dg-coated beads to Raji cells. Used as antigen for immunization, it allowed the efficient and well-aimed generation of antisera which specifically blocked attachment of C3dg-coated beads to Raji B cells. Thus, insect cell derived rsCR2.1-4 has proved a valuable tool to study the functional domain of CR2 and its immunoregulatory capacity in B-lymphocytes.
- Published
- 1997
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