Expression and characterization of recombinant soluble porcine CD3 ectodomain molecules: Mapping the epitope of an anti-porcine CD3 monoclonal antibody 898H2-6-15

► Porcine CD3 ectodomain molecules were expressed in Escherichia coli and refolded in vitro. ► The refolded porcine CD3 ectodomain molecules were purified with a strong anion exchange resin. ► Porcine CD3εγ ectodomain single-chain fusion protein can bind to an anti-porcine CD3 mAb 898H2-6-15. The porcine CD3 specific monoclonal antibody 898H2-6-15 has been used in allo- and xeno-transplantation studies as a porcine CD3 marker and as an effective T cell depletion reagent when conjugated to the diphtheria toxin mutant, CRM9. A recombinant anti-porcine CD3 immuntoxin was recently developed using single-chain variable fragments (scFv) derived from 898H2-6-15. In this study, using published sequence data, we have expressed the porcine CD3 ectodomain molecules in E. coli through inclusion body isolation and in vitro refolding approach. The expressed and refolded porcine CD3 ectodomain molecules include CD3ε, CD3γ, CD3δ, CD3εγ heterodimer, CD3εδ heterodimer, CD3εγ single-chain fusion protein and CD3εδ single-chain fusion protein. These refolded porcine CD3 ectodomain molecules were purified with a strong anion exchange resin Poros 50HQ. ELISA analysis demonstrated that only the porcine CD3εγ ectodomain single-chain fusion protein can bind to the porcine CD3 specific monoclonal antibody 898H2-6-15. The availability of this porcine CD3εγ ectodomain single-chain fusion protein will allow screening for affinity matured variants of scFv derived from 898H2-6-15 to improve the recombinant anti-porcine CD3 immunotoxin. Porcine CD3εγ ectodomain single-chain fusion protein will also be a very useful reagent to study the soluble phase interaction between porcine CD3εγ and porcine CD3 antibodies such as 898H2-6-15.