RESUMO
Optimizing antibody purification is crucial to overcoming a bottleneck in the costly manufacturing process for antibody therapy. To address this issue, we designed a pH-sensitive Staphylococcus aureus protein A variant that retained its innate stability and affinity toward antibody. On the basis of structural information and mutation analysis data, we identified candidate positions for accumulative histidine substitutions to cause electrostatic repulsion under acidic conditions. The histidine substitutions effectively decreased the dissociation rate under acidic conditions by three orders of magnitude. Avoiding deleterious effects of the substitutions, we successfully engineered a protein A variant that exhibited high pH sensitivity and maintained affinity, thermal stability, and alkaline tolerance. The variant was capable of serving as an affinity ligand that made affinity chromatography under milder acidic conditions possible; the elution peak shifted from pH 4.2 to 5.6. Only two substitutions were needed to achieve this pH sensitivity. This structure-based approach is applicable to other protein-based ligands.
Assuntos
Histidina/química , Proteína Estafilocócica A/química , Substituição de Aminoácidos , Cromatografia de Afinidade , Concentração de Íons de Hidrogênio , Engenharia de Proteínas , Ressonância de Plasmônio de SuperfícieRESUMO
Protein-protein interaction in response to environmental conditions enables sophisticated biological and biotechnological processes. Aiming toward the rational design of a pH-sensitive protein-protein interaction, we engineered pH-sensitive mutants of streptococcal protein G B1, a binder to the IgG constant region. We systematically introduced histidine residues into the binding interface to cause electrostatic repulsion on the basis of a rigid body model. Exquisite pH sensitivity of this interaction was confirmed by surface plasmon resonance and affinity chromatography employing a clinically used human IgG. The pH-sensitive mechanism of the interaction was analyzed and evaluated from kinetic, thermodynamic, and structural viewpoints. Histidine-mediated electrostatic repulsion resulted in significant loss of exothermic heat of the binding that decreased the affinity only at acidic conditions, thereby improving the pH sensitivity. The reduced binding energy was partly recovered by "enthalpy-entropy compensation." Crystal structures of the designed mutants confirmed the validity of the rigid body model on which the effective electrostatic repulsion was based. Moreover, our data suggested that the entropy gain involved exclusion of water molecules solvated in a space formed by the introduced histidine and adjacent tryptophan residue. Our findings concerning the mechanism of histidine-introduced interactions will provide a guideline for the rational design of pH-sensitive protein-protein recognition.
Assuntos
Anticorpos Monoclonais/química , Proteínas de Bactérias/química , Fragmentos Fc das Imunoglobulinas/química , Imunoglobulina G/química , Modelos Moleculares , Streptococcus/química , Anticorpos Monoclonais/metabolismo , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Histidina/química , Histidina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoglobulina G/metabolismo , Cinética , Ligação Proteica , Eletricidade Estática , Ressonância de Plasmônio de Superfície , Triptofano/química , Triptofano/metabolismoRESUMO
8-hydroxy-2'-deoxyguanosine (8-OHdG), as a measure of oxidative stress, was measured in healthy Japanese volunteers using an ELISA (New 8-OHdG Check, JICA). Analysis of daytime spot urine of 83 healthy male subjects and smoking habit, exercise and age revealed significant correlation only between the urinary level of 8-OHdG and age. As the inter-individual variation of 8-OHdG of the daytime spot urine was relatively high, we next determined inter-and intra-individual variation of 5 healthy volunteers. The levels of 8-OHdG/creatinine in morning spot urine significantly correlated with 8-OHdG levels in 24-h pool urine. Thus, a morning spot urine sample can be used for the measurement of 8-OHdG instead of inconvenient 24-h sampling.