Affinity immobilization of a genetically engineered bifunctional hybrid protein.
Protein A from Staphylococcus aureus (SpA) is a receptor for the Fc domain of several classes of antibodies including immunoglobin G (IgG). A hybrid protein consisting of protein A and the enzyme beta-lactamase has been constructed using recombinant DNA techniques. The functional characteristics of the hybrid protein adsorbed on IgG-coated Sepharose matrices were studied in detail and compared to those of (i) the hybrid protein in solution and (ii) beta-lactamase covalently immobilized on CNBr-activated Sepharose. Protein A--beta-lactamase bound tightly and specifically to IgG-Sepharose and could be stored for at least 4 weeks without dissociation. The rate of penicillin G hydrolysis by the beta-lactamase domain of the immobilized hybrid protein was found to depend on the amount of IgG covalently coupled to the support. For all IgG loads, higher specific activities and lower Km values relative to covalently immobilized beta-lactamase were obtained. Adsorption of the hybrid protein on the support resulted in increased stability to thermal deactivation. These results indicate that bifunctional hybrid proteins can be useful for the affinity immobilization of enzymes.
Baneyx
F
F
Department of Chemical Engineering, University of Texas, Austin 78712.
Schmidt
C
C
Georgiou
G
G
D002846:Chromatography, Affinity / Q000379:methods; D004800:Enzymes, Immobilized / Q000378:metabolism; D005818:Genetic Engineering; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G; D007700:Kinetics; D010400:Penicillin G / Q000378:metabolism; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification*; D013205:Staphylococcal Protein A / Q000235:genetics* / Q000302:isolation & purification; D013211:Staphylococcus aureus / Q000235:genetics; D014508:Urea / Q000494:pharmacology; D001618:beta-Lactamases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D004800:Enzymes, Immobilized; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D014508:Urea; D001618:beta-Lactamases; D010400:Penicillin G