RESUMEN
In antigen-antibody interactions, the high avidity of antibodies depends on the affinity and number of the individual binding sites. To develop artificial antibodies with multiple valency, we have fused the single-chain antibody Fv fragments to core streptavidin. The resulting fusion protein, termed scFv::strep, was found after expression in Escherichia coli in periplasmic inclusion bodies. After purification of the recombinant product by immobilized metal affinity chromatography, refolding and size-exclusion FPLC, tetrameric complexes resembling those of mature streptavidin were formed. The purified tetrameric scFv::strep complexes demonstrated both antigen- and biotin-binding activity, were stable over a wide range of pH and did not dissociate at high temperatures (up to 70 degrees C). Surface plasmon resonance measurements in a BIAlite system showed that the pure scFv::strep tetramers bound immobilized antigen very tightly and no dissociation was measurable. The association rate constant for scFv::strep tetramers was higher than those for scFv monomers and dimers. This was also reflected in the apparent constants, which was found to be 35 times higher for pure scFv::strep tetramers than monomeric single-chain antibodies. We could also show that most of biotin binding sites were accessible and not blocked by biotinylated E.coli proteins or free biotin from the medium. These sites should therefore facilitate the construction of bispecific multivalent antibodies by the addition of biotinylated ligands.
Asunto(s)
Proteínas Bacterianas/genética , Fragmentos de Inmunoglobulinas/inmunología , Región Variable de Inmunoglobulina/inmunología , Proteínas Recombinantes de Fusión/inmunología , Anticuerpos/química , Anticuerpos/genética , Anticuerpos/metabolismo , Antígenos/metabolismo , Proteínas Bacterianas/inmunología , Sitios de Unión/genética , Biotina/metabolismo , Western Blotting , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Expresión Génica/genética , Guanidina , Guanidinas/farmacología , Concentración de Iones de Hidrógeno , Fragmentos de Inmunoglobulinas/genética , Región Variable de Inmunoglobulina/genética , Cinética , Plásmidos/genética , Unión Proteica , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Estreptavidina , TemperaturaRESUMEN
To facilitate the isolation of IgG antibody Fv-DNA sequences from hybridoma cell lines, we have established a polymerase chain reaction (PCR) procedure requiring only a small number of primers. The sense primers homologous to DNA coding for the first framework sequences were designed to hybridize to all the known antibody sequences under conditions that permit a high number of mismatches. The antisense primers were homologous to DNA coding for the beginning of the constant regions of the gamma and kappa chains. Restriction sites introduced by the primers enable the DNA to be cloned into bacterial expression vectors. Only three sense VH primers and two sense VL primers paired with one backward primer for the heavy and light chains, respectively, were necessary for the amplification of Fv-DNA from a total of 17 rodent cell lines that we have so far worked with. These consisted of 12 mouse cell lines and five rat cell lines. This procedure will therefore probably be sufficient to isolate the Fv-DNA from most mouse cell lines and possibly also from most rat cell lines.
