Heterodimeric CD3ϵγ extracellular domain fragments: production, purification and structural analysis

Abstract

The CD3 polypeptides (ϵ, γ, and δ) are non-covalently associated signaling subunits of the T cell receptor which form non-disulfide linked ϵγ and ϵδ heterodimers. With the goal of investigating their structure, Escherichia coli expression was utilized to produce CD3 ectodomain fragments including the murine CD3ϵ subunit N-terminal Ig-like extracellular domain alone or as a single chain construct with that of CD3γ. The latter links the CD3γ segment to the C terminus of the CD3ϵ segment via a 26 amino acid peptide (scCD3ϵγ26). Although CD3ϵ could be produced at high yield when directed to inclusion bodies, the refolded monomeric CD3ϵ was not native as judged by monoclonal antibody binding using surface plasmon resonance and was largely unstructured by 15N- 1H two-dimensional NMR analysis. In contrast, scCD3ϵγ26 could be refolded readily into a native state as shown by CD, NMR and mAb reactivity. The linker length between CD3ϵ and CD3γ is critical since scCD3ϵγ16 containing a 16 residue connector failed to generate a stable heterodimer. Collectively, the results demonstrate that: (i) soluble heterodimeric fragments of CD3 can be produced; (ii) cotranslation of CD3 chains insures proper folding even in the absence of the conserved ectodomain stalk region (CxxCxE); and (iii) CD3ϵ has a more stable tertiary protein fold than CD3γ.