Assuntos
Complexo Antígeno-Anticorpo/isolamento & purificação , Proteína Estafilocócica A/metabolismo , Sequência de Aminoácidos , Complexo Antígeno-Anticorpo/sangue , Complexo Antígeno-Anticorpo/metabolismo , Sítios de Ligação , Fenômenos Biofísicos , Biofísica , Humanos , Imunoglobulina G/metabolismo , Técnicas In Vitro , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Engenharia de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteína Estafilocócica A/químicaRESUMO
The functional antigen binding region of antidinitrophenol mouse IgA myeloma MOPC 315 has been produced as a single-chain Fv (sFv) protein in E. coli. Recombinant 315 proteins included sFv alone, a bifunctional fusion protein with amino-terminal fragment B (FB) of staphylococcal protein A, and a two-chain 315 Fv fragment. Successful refolding of the 315 sFv required formation of disulfide bonds while the polypeptide was in a denatured state, as previously observed for the parent Fv fragment. Affinity-purified recombinant 315 proteins showed full recovery of specific activity, with values for Ka,app of 1.5 to 2.2 x 10(6) M-1, equivalent to the parent 315 Fv fragment. As observed for natural 315 Fv, the sFv region of active FB-sFv315 fusion protein was resistant to pepsin treatment, whereas inactive protein was readily degraded. These experiments will allow the application of protein engineering to the 315 single-chain Fv; such studies can advance structure-function studies of antibody combining sites and lead to an improved understanding of single-chain Fv proteins.
