title: Optimization of the antibody C(H)3 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(H)3 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(H)3. Under identical conditions, expression of bovine C(H)3 resulted consistently in the highest yields of properly folded/oxidized protein. Circular dichroism and fluorescence experiments demonstrate that oxidized bovine and murine C(H)3 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(H)3. Combination of three of these mutants increased the thermal stability of the molecule to 86 degrees 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(H)3 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.
authors.affiliation: Torrey Mesa Research Institute, 3115 Merryfield Row, San Diego, CA 92130, USA. stephen.demarest@diversa.com
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Demarest
authors.forename: Jeff
authors.initials: J
authors.lastname: Rogers
authors.forename: Geneviève
authors.initials: G
authors.lastname: Hansen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015195:Drug Design; D004355:Drug Stability; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry* / Q000235:genetics; D007074:Immunoglobulin G / Q000737:chemistry* / Q000235:genetics; D007143:Immunoglobulin Heavy Chains; D015202:Protein Engineering; D017434:Protein Structure, Tertiary
chemical_list: D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D007143:Immunoglobulin Heavy Chains