RESUMO
When panned with a transient heat denaturation approach against target enzymes, a human V(H) (antibody heavy chain variable domain) phage display library yielded V(H)s with composite characteristics of binding, non-aggregation and reversible thermal unfolding. Moreover, selection was characterized by enrichment for V(H)s with (i) an even number of disulfide forming Cys residues in complementarity-determining region (CDR) 1 and CDR3 and (ii) acidic isoelectric points. This parallels naturally occurring camelid and shark single-domain antibodies (sdAbs) which are also characterized by (i) solubility and reversible unfolding, (ii) a high occurrence of disulfide forming Cys in their CDRs, particularly, in CDR1 and CDR3 and (iii) acidic V(H)s as inferred here by a pI distribution analysis, reported here, of pools of human and camelid V(H) and V(H)H (camelid heavy chain antibody V(H)) sequences. Our results, reinforced by previous observations by others, suggest that protein acidification may yet be another mechanism nature has devised to create functional sdAbs and that this concept along with the inclusion of inter-CDR disulfide linkages may be applied to human V(H) domains/libraries for non-aggregation optimization. In addition, calculation of theoretical pIs of V(H)s selected by panning may be used for rapid and precise identification of non-aggregating V(H)s.
Assuntos
Evolução Molecular Direcionada , Dissulfetos/metabolismo , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/genética , Animais , Camelídeos Americanos , Distribuição de Qui-Quadrado , Regiões Determinantes de Complementaridade/genética , Humanos , Concentração de Íons de Hidrogênio , Cadeias Pesadas de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/metabolismo , Ponto Isoelétrico , Biblioteca de Peptídeos , Plasmídeos/genética , Desnaturação Proteica , TemperaturaRESUMO
Evidence is provided that dromedary heavy-chain antibodies, in vivo-matured in the absence of light chains, are a unique source of inhibitory antibodies. After immunization of a dromedary with bovine erythrocyte carbonic anhydrase and porcine pancreatic alpha-amylase, it was demonstrated that a considerable amount of heavy-chain antibodies, acting as true competitive inhibitors, circulate in the bloodstream. In contrast, the conventional antibodies apparently do not interact with the enzyme's active site. Next we illustrated that peripheral blood lymphocytes are suitable for one-step cloning of the variable domain fragments in a phage-display vector. By bio-panning, several antigen-specific single-domain fragments are readily isolated for both enzymes. In addition we show that among those isolated fragments active site binders are well represented. When produced as recombinant protein in Escherichia coli, these active site binders appear to be potent enzyme inhibitors when tested in chromogenic assays. The low complexity of the antigen-binding site of these single-domain antibodies composed of only three loops could be valuable for designing smaller synthetic inhibitors.
Assuntos
Inibidores da Anidrase Carbônica , Inibidores Enzimáticos/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/imunologia , alfa-Amilases/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Afinidade de Anticorpos , Especificidade de Anticorpos , Camelus , Anidrases Carbônicas/imunologia , Anidrases Carbônicas/metabolismo , Bovinos , Inibidores Enzimáticos/isolamento & purificação , Humanos , Cadeias Pesadas de Imunoglobulinas/classificação , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/isolamento & purificação , Região Variável de Imunoglobulina/classificação , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/isolamento & purificação , Masculino , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão , Alinhamento de Sequência , Suínos , alfa-Amilases/imunologiaRESUMO
Functional heavy-chain gamma-immunoglobulins lacking light chains occur naturally in Camelidae. We now show the feasibility of immunising a dromedary, cloning the repertoire of the variable domains of its heavy-chain antibodies and panning, leading to the successful identification of minimum sized antigen binders. The recombinant binders are expressed well in E. coli, extremely stable, highly soluble, and react specifically and with high affinity to the antigens. This approach can be viewed as a general route to obtain small binders with favourable characteristics and valuable perspectives as modular building blocks to manufacture multispecific or multifunctional chimaeric proteins.