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 insufficient for structural studies. To circumvent these problems we established an indirect immunoaffinity chromatography method based on engineered Fv fragments. cDNAs encoding the variable domains of hybridoma-derived antibodies raised against various membrane proteins were cloned and expressed in Escherichia coli. The Fv fragments were engineered to serve as bifunctional adaptor molecules. The Fv fragment binds to the epitope of the membrane protein, while the Strep tag affinity peptide, which was fused to the carboxy-terminus of the VH chain, immobilizes the antigen-Fv complex on a streptavidin sepharose column. The usefulness of this technique is illustrated with membrane protein complexes from Paracoccus denitrificans, namely, the cytochrome c oxidase (EC 1.9.3.1), the ubiquinol:cytochrome c oxidoreductase (EC 1.10.2.2), and 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 streptavidin affinity chromatography. Pure and highly active membrane protein complexes were eluted in the Fv-bound form using diaminobiotin for mild competitive displacement of the Strep tag. The affinity column could thus be reused under continuous operation for several months. Five to 10 mg of membrane protein complexes could be obtained without any detectable impurities within five hours.
Kleymann
G
G
Max-Planck-Institut für Biophysik, Abteilung Molekulare Membranbiologie, Frankfurt, Germany.
Ostermeier
C
C
Ludwig
B
B
Skerra
A
A
Michel
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D014450:Electron Transport Complex III / Q000302:isolation & purification; D003576:Electron Transport Complex IV / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006825:Hybridomas / Q000276:immunology; D007128:Immunoglobulin Fragments / Q000235:genetics*; D007158:Immunologic Techniques; D007202:Indicators and Reagents; D008565:Membrane Proteins / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010231:Paracoccus denitrificans / Q000737:chemistry; D015202:Protein Engineering; D019809:Streptavidin
D007128:Immunoglobulin Fragments; D007202:Indicators and Reagents; D008565:Membrane Proteins; C055966:immunoglobulin Fv; D019809:Streptavidin; D003576:Electron Transport Complex IV; D014450:Electron Transport Complex III