Expression and characterization of recombinant soluble monkey CD3 molecules: mapping the FN18 polymorphic epitope.
The monoclonal antibody FN18 has been used as a marker for monkey T cells and as a T cell depleting reagent when conjugated to binding site mutants of diphtheria toxin. This anti-CD3 antibody shares certain properties with its anti-human counterparts UCHT1, OKT3 and Leu-4 in that it precipitates two different CD3 chains from membrane detergent extracts. However, in contrast to human CD3, rhesus and cynomolgus monkeys display CD3 polymorphisms producing FN18 negative phenotypes. Using recently published sequence data, we have expressed the ectodomains of cynomolgus CD3-epsilon CD3-gamma and CD-delta chains in E. coli, and have refolded these chains separately and in pairs to produce CD3 homo- and heterodimeric proteins. These proteins were fractionated by anion exchange according to their differing isoelectric points and further identified by size differences on SDS gels. On the basis of ELISA, the FN18 epitope is restricted to the CD3-epsilongamma ectodomain heterodimer, CD-epsilondelta and CD-epsilonepsilon being non-reactive. Either of the two amino acid polymorphisms reported in the CD3-epsilon chain were sufficient to degrade the bioactivity of the CD3-epsilongamma towards FN18.
Wang
Zhirui
Z
Section of Biophysical Chemistry, Laboratory of Molecular Biology, National Institute of Mental Health, Building 36, Room 1B08, 36 Convent Drive, Bethesda, MD 28092-4034, USA.
Neville
David M
DM
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000378:metabolism*; D017252:CD3 Complex / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D004926:Escherichia coli / Q000276:immunology; D007519:Isoantigens / Q000276:immunology*; D007526:Isoelectric Point; D008252:Macaca fascicularis / Q000276:immunology; D008970:Molecular Weight; D009154:Mutation; D011110:Polymorphism, Genetic; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism*; D012995:Solubility
D000911:Antibodies, Monoclonal; D017252:CD3 Complex; D007519:Isoantigens; D011994:Recombinant Proteins