Expression and purification of antigenically active soluble derivatives of the heterodimeric and homodimeric forms of the mouse CD8 lymphocyte membrane glycoprotein

The T lymphocyte membrane glycoprotein CD8 enhances antigen recognition by class I-restricted T cells. There are two naturally occurring forms of CD8, an αβ heterodimer expressed by the majority of CD8 + T cells, and a less abundant αα homodimer found on specialised T cell subsets. An expression strategy was developed for production of soluble CD8αα and CD8αβ extracellular domains for use in ligand binding studies. Mouse CD8α was expressed autonomously as a homodimer at 10 mg/l in mammalian fibroblasts, but CD8β was not expressed at significant levels in the absence of CD8α. Co-expression with CD8α led to significant enhancement in the level of CD8β expression, which was secreted as a non-covalent heterodimer at 3 mg/l with CD8α. Despite the marked increase of CD8β expression in the presence of CD8α, an excess of soluble CD8αα homodimer was also present in the supernatant of co-expressing cell clones. In order to resolve the CD8αα homodimer from the CD8αβ heterodimer, affinity chromatographic techniques specific for the CD8β subunit were employed. Purification procedures requiring elution from affinity matrices at low pH led to substantial losses in the total antigenic activity and partial subunit dissociation of the soluble CD8αβ heterodimer. The inclusion of a hexahistidine tag at the C-terminus of CD8β enabled affinity purification of soluble CD8αβ (and sCD8αα) under neutral conditions, yielding recombinant protein with the correct stoichiometry and full antigenic activity. This method may prove useful for production of other soluble recombinant heterodimeric receptor proteins whose antigenicity is affected by denaturation during immunoaffinity purification.