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

The CD3 polypeptides (epsilon, gamma, and delta) are non-covalently associated signaling subunits of the T cell receptor which form non-disulfide linked epsilon gamma and epsilon delta heterodimers. With the goal of investigating their structure, Escherichia coli expression was utilized to produce CD3 ectodomain fragments including the murine CD3 epsilon subunit N-terminal Ig-like extracellullar domain alone or as a single chain construct with that of CD3 gamma. The latter links the CD3 gamma segment to the C terminus of the CD3 epsilon segment via a 26 amino acid peptide (scCD3 epsilon gamma 26). Although CD3 epsilon could be produced at high yield when directed to inclusion bodies, the refolded monomeric CD3 epsilon was not native as judged by monoclonal antibody binding using surface plasmon resonance and was largely unstructured by N-15-H-1 two-dimensional NMR analysis. In contrast, scCD3 epsilon gamma 26 could be refolded readily into a native state as shown by CD, NMR and mAb reactivity. The linker length between CD3 epsilon and CD3 gamma is critical since scCD3 epsilon gamma 16 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) CD3 epsilon has a more stable tertiary protein fold than CD3 gamma. (C) 2000 Academic Press.