ENGINEERED FV FRAGMENTS AS A TOOL FOR THE ONE-STEP PURIFICATION OF INTEGRAL MULTISUBUNIT MEMBRANE-PROTEIN COMPLEXES

The preparation of pure and homogeneous membrane proteins or membrane protein complexes is time consuming, and the yields are frequently ins ufficient for structural studies. To circumvent these problems we esta blished an indirect immunoaffinity chromatography method based on engi neered Fv fragments. cDNAs encoding the variable domains of hybridoma- derived antibodies raised against various membrane proteins were clone d and expressed in Escherichia coli. The Fv fragments were engineered to serve as bifunctional adaptor molecules. The Fv fragment binds to t he epitope of the membrane protein, while the Strep tag affinity pepti de, which was fused to the carboxy-terminus of the V-H chain, immobili zes the antigen-Fv complex on a streptavidin sepharose column. The use fulness of this technique is illustrated with membrane protein complex es from Paracoccus denitrificans, namely, the cytochrome c oxidase (EC 1.9.3.1), the ubiquinol:cytochrome c oxidoreductase (EC 1.10.2.2), an d subcomplexes or individual subunits thereof. These membrane proteins were purified simply by combining the crude P. denitrificans membrane preparation with the E. coli periplasmic cell fraction containing the corresponding Fv fragment, followed by solubilization and streptavidi n affinity chromatography. Pure and highly active membrane protein com plexes were eluted in the Fv-bound form using diaminobiotin for mild c ompetitive displacement of the Strep tag. The affinity column could th us be reused under continuous operation for several months. Five to 10 mg of membrane protein complexes could be obtained without any detect able impurities within five hours.