Heterodimeric CD3epsilongamma 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 epsilongamma and epsilondelta heterodimers. With the goal of investigating their structure, Escherichia coli expression was utilized to produce CD3 ectodomain fragments including the murine CD3epsilon subunit N-terminal Ig-like extracellular domain alone or as a single chain construct with that of CD3gamma. The latter links the CD3gamma segment to the C terminus of the CD3epsilon segment via a 26 amino acid peptide (scCD3epsilongamma26). Although CD3epsilon could be produced at high yield when directed to inclusion bodies, the refolded monomeric CD3epsilon was not native as judged by monoclonal antibody binding using surface plasmon resonance and was largely unstructured by (15)N-(1)H two-dimensional NMR analysis. In contrast, scCD3epsilongamma26 could be refolded readily into a native state as shown by CD, NMR and mAb reactivity. The linker length between CD3epsilon and CD3gamma is critical since scCD3epsilongamma16 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) CD3epsilon has a more stable tertiary protein fold than CD3gamma.
Kim
K S
KS
Laboratory of Immunobiology, Dana-Farber Cancer Institute and Department of Medicine, Boston, MA, 02115, USA.
Sun
Z Y
ZY
Wagner
G
G
Reinherz
E L
EL
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D017252:CD3 Complex / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D002942:Circular Dichroism; D017124:Conserved Sequence / Q000235:genetics; D019281:Dimerization; D004926:Escherichia coli; D005091:Exons / Q000235:genetics; D007136:Immunoglobulins / Q000737:chemistry; D002479:Inclusion Bodies / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D016415:Sequence Alignment; D020349:Surface Plasmon Resonance
D000911:Antibodies, Monoclonal; D017252:CD3 Complex; D007136:Immunoglobulins; D010446:Peptide Fragments; D021122:Protein Subunits; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins