Expression in insect cells of the functional domain of CD21 (complement receptor type two) as a truncated soluble molecule using a baculovirus vector

Immunopharmacology. 1997 Dec;38(1-2):141-8. doi: 10.1016/s0162-3109(97)00062-3.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Monoclonal / genetics
  • Antibodies, Monoclonal / immunology
  • Antigen-Antibody Reactions
  • B-Lymphocytes / immunology
  • B-Lymphocytes / metabolism
  • Baculoviridae / genetics*
  • Female
  • Genetic Vectors / genetics*
  • Genetic Vectors / immunology
  • Humans
  • Immunization
  • Mice
  • Mice, Inbred BALB C
  • Plasmids / genetics*
  • Plasmids / immunology
  • Receptors, Complement / genetics*
  • Receptors, Complement / immunology
  • Receptors, Complement 3d / biosynthesis*
  • Receptors, Complement 3d / immunology
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology
  • Spodoptera

Substances

  • Antibodies, Monoclonal
  • Receptors, Complement
  • Receptors, Complement 3d
  • Recombinant Proteins