title: Expression and purification of antigenically active soluble derivatives of the heterodimeric and homodimeric forms of the mouse CD8 lymphocyte membrane glycoprotein.
abstract: The T lymphocyte membrane glycoprotein CD8 enhances antigen recognition by class I-restricted T cells. There are two naturally occurring forms of CD8, an alphabeta heterodimer expressed by the majority of CD8(+) T cells, and a less abundant alphaalpha homodimer found on specialised T cell subsets. An expression strategy was developed for production of soluble CD8alphaalpha and CD8alphabeta extracellular domains for use in ligand binding studies. Mouse CD8alpha was expressed autonomously as a homodimer at 10 mg/l in mammalian fibroblasts, but CD8beta was not expressed at significant levels in the absence of CD8alpha. Co-expression with CD8alpha led to significant enhancement in the level of CD8beta expression, which was secreted as a non-covalent heterodimer at 3 mg/l with CD8alpha. Despite the marked increase of CD8beta expression in the presence of CD8alpha, an excess of soluble CD8alphaalpha homodimer was also present in the supernatant of co-expressing cell clones. In order to resolve the CD8alphaalpha homodimer from the CD8alphabeta heterodimer, affinity chromatographic techniques specific for the CD8beta 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 CD8alphabeta heterodimer. The inclusion of a hexahistidine tag at the C-terminus of CD8beta enabled affinity purification of soluble CD8alphabeta (and sCD8alphaalpha) 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.
authors: Department of Pathology and Immunology, Monash Medical School, Commercial Road, Vic. 3181, Prahran, Australia.
authors: D G
authors: DG
authors: Pellicci
authors: A A
authors: AA
authors: Kortt
authors: L G
authors: LG
authors: Sparrow
authors: P J
authors: PJ
authors: Hudson
authors: H V
authors: HV
authors: Sorensen
authors: S J
authors: SJ
authors: Davis
authors: B J
authors: BJ
authors: Classon
mesh_terms: D000818:Animals; D016827:CD8 Antigens / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D016466:CHO Cells / Q000378:metabolism; D002846:Chromatography, Affinity; D006224:Cricetinae; D019281:Dimerization; D051379:Mice; D020033:Protein Isoforms / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D051381:Rats; D012995:Solubility
chemical_list: D016827:CD8 Antigens; C409469:CD8 antigen, alpha chain; D020033:Protein Isoforms