Optimization of the Antibody CH3 Domain by Residue Frequency Analysis of IgG Sequences

In an attempt to enhance the overall assembly, yield and half-life of recombinant antibody proteins, we have cloned and expressed several IgG1 CH3 domains and examined their folding/refolding characteristics. We utilized a cytoplasmic bacterial expression system with a thioredoxin reductase knock-out strain of BL21(DE3) to produce bovine, murine and human CH3. Under identical conditions, expression of bovine CH3 resulted consistently in the highest yields of properly folded/oxidized protein. Circular dichroism and fluorescence experiments demonstrate that oxidized bovine and murine CH3 have surprisingly similar structures and stabilities, considering the marginal sequence conservation between the two molecules. Residue frequency analysis using a limited data set of 36 unique Fc sequences originating from 19 different mammalian species targeted five specific sites for optimization within bovine CH3. Combination of three of these mutants increased the thermal stability of the molecule to 86 °C. Comparison of this approach to similar studies using larger sequence databases and/or different selection criteria suggests sequence database design can increase the success rate for identifying residue sites worth optimizing. This optimized CH3 domain can be used as a particularly stable platform for functional design and can be grafted into full-length antibody sequences to enhance their thermodynamic parameters and shelf-life.