The immunoglobulin (Ig) constant CH2 domain is critical for antibody effector functions. Isolated CH2 domains are promising scaffolds for construction of libraries containing diverse binders that could also confer some effector functions. We have previously shown (Gong et al., JBC 2009) that an isolated human CH2 domain is relatively unstable to thermally induced unfolding but its stability can be improved by engineering an additional disulfide bond. We have hypothesized that the stability of this engineered antibody domain can be further increased by removing unstructured residues. To test our hypothesis we removed the seven N-terminal residues that are in a random coil as suggested by our analysis of the isolated CH2 crystal structure and NMR data. The resulting shortened engineered CH2 (m01s) was highly soluble, monomeric and remarkably stable with a melting temperature (Tm) of 82.6ºC which is about 10ºC and 30ºC higher than that of the original stabilized CH2 (m01) and CH2, respectively. m01s and m01 were more resistant to protease digestion than CH2. A newly identified anti-CH2 antibody which recognizes a conformational epitope bound to m01s significantly better (>10-fold higher affinity) than to CH2 and slightly better than to m01. m01s bound to a recombinant soluble human Fc neonatal receptor at pH 6.0 more strongly than CH2. These data suggest that shortening
of the m01 N-terminus significantly increases stability without disrupting its conformation, and that our approach for increasing stability and decreasing size by removing unstructured regions may also apply to other proteins.