Single antibody domains as small recognition units: design and in vitro antigen selection of camelized, human VH domains with improved protein stability.
Folding stabilities of camelized human antibody VH domains were studied through the determination of their melting points in thermodenaturation experiments. The melting point of a VH domain originating from a synthetic library of human VHs, which had been optimized for the use as small recognition units through the mimicking of camelid antibody heavy chains occurring naturally without light chain, was 56.6 degrees C compared with 71.2 degrees C of the original human VH. Its stability was improved (melting point 61.6 degrees C) through three mutations to mimic camelid VHs even further: Va137 was replaced by phenylalanine and two cysteines were introduced at position 33 and 100b. The resulting VH folded properly and formed a second intradomain disulphide between the extra cysteines. The new mutations were then built constitutively into a phage-display VH library, from which antigen-specific VHs were selected. Two were analysed for stability with melting points of 72.6 and 75.3 degrees C. Thus secondary camelization enabled the isolation of VHs with improved folding stabilities exceeding even that of the original human VH. This indicates an effect on folding stability for some mutations specific in the light chain lacking camelid heavy chains.
Davies
J
J
MRC Laboratory of Molecular Biology, Cambridge, UK.
Riechmann
L
L
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D002162:Camelus / Q000235:genetics / Q000276:immunology*; D003553:Cystine / Q000032:analysis; D004797:Enzyme-Linked Immunosorbent Assay; D015723:Gene Library; D005803:Genes, Immunoglobulin; D006358:Hot Temperature; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D003553:Cystine