Optimization of the Antibody C H3 Domain by Residue Frequency Analysis of IgG Sequences

Abstract

In an attempt to enhance the overall assembly, yield and half-life of recombinant antibody proteins, we have cloned and expressed several IgG1 C H3 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 C H3. Under identical conditions, expression of bovine C H3 resulted consistently in the highest yields of properly folded/oxidized protein. Circular dichroism and fluorescence experiments demonstrate that oxidized bovine and murine C H3 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 C H3. 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 C H3 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.