PMID: 9634756Feb 1, 1995Paper

Engineered Fv fragments as a tool for the one-step purification of integral multisubunit membrane protein complexes

Bio/technology
G KleymannHartmut Michel

Abstract

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...Continue Reading

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Citations

Feb 1, 1996·Journal of Bioenergetics and Biomembranes·R M GaravitoP J Loll
Aug 7, 2002·Current Opinion in Structural Biology·Carola Hunte, Hartmut Michel
Nov 5, 1997·Current Opinion in Structural Biology·C Ostermeier, H Michel
May 5, 2000·Biomolecular Engineering·A Skerra, T G Schmidt
Mar 4, 2011·Proceedings of the National Academy of Sciences of the United States of America·Iris von der HochtHartmut Michel
Jan 13, 2012·The Journal of Biological Chemistry·Kristina KirchbergUlrike Alexiev
Apr 2, 1998·Hybridoma·N B BlattG D Glick
Dec 1, 1995·European Journal of Biochemistry·A WarneM Saraste
Oct 6, 1997·Proceedings of the National Academy of Sciences of the United States of America·C OstermeierH Michel
Apr 26, 2000·Proceedings of the National Academy of Sciences of the United States of America·M RuitenbergK Fendler
Aug 1, 1995·Quarterly Reviews of Biophysics·R Grisshammer, C G Tate
Feb 8, 2006·FEBS Letters·Fraser MacMillanHartmut Michel
Jun 3, 2015·Biotechnology and Bioengineering·Conor FieldsGil U Lee
Oct 27, 2007·Protein Science : a Publication of the Protein Society·Sehat NauliJames U Bowie
May 7, 2015·Acta Crystallographica. Section D, Biological Crystallography·Martin KohlstaedtHartmut Michel
Nov 5, 1997·Protein Expression and Purification·J NilssonP A Nygren
Jul 9, 2002·Biochimica Et Biophysica Acta·Christian K EngelGilbert G Privé
Nov 25, 2003·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Yasuhiro Kashino
Apr 9, 2015·ACS Applied Materials & Interfaces·Ning-Ning LuWei-Hua Huang

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