Engineered Fv Fragments as a Tool for the One-Step Purification of Integral Multisubunit Membrane Protein Complexes

Kleymann, Gerald; Ostermeier, Christian; Ludwig, Bernd; Skerra, Arne; Michel, Hartmut

doi:10.1038/nbt0295-155

Cited by 95 publications

(77 citation statements)

References 39 publications

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“…Later, the gene for subunit III was discovered (15) and a ''three-subunit enzyme'' was isolated subsequently with dodecyl-␤-D-maltoside as detergent (16). Recently, it was recognized that an additional small subunit, subunit IV, was present in such preparations (17,18).…”

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confidence: 99%

Structure at 2.7 Å resolution of the Paracoccus denitrificans two-subunit cytochrome c oxidase complexed with an antibody F _V fragment

Ostermeier

Harrenga

Ermler

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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The aa 3 type cytochrome c oxidase consisting of the core subunits I and II only was isolated from the soil bacterium Paracoccus denitrificans and crystallized as complex with a monoclonal antibody F v fragment. Crystals could be grown in the presence of a number of different nonionic detergents. However, only undecyl-␤-D-maltoside and cyclohexyl-hexyl-␤-D-maltoside yielded well-ordered crystals suitable for high resolution x-ray crystallographic studies. The crystals belong to space group P2 1 2 1 2 1 and diffract x-rays to at least 2.5 Å (1 Å ‫؍‬ 0.1 nm) resolution using synchrotron radiation. The structure was determined to a resolution of 2.7 Å using molecular replacement and refined to a crystallographic R-factor of 20.5% (R free ‫؍‬ 25.9%). The refined model includes subunits I and II and the 2 chains of the F v fragment, 2 heme A molecules, 3 copper atoms, and 1 Mg͞Mn atom, a new metal (Ca) binding site, 52 tentatively identified water molecules, and 9 detergent molecules. Only four of the water molecules are located in the cytoplasmic half of cytochrome c oxidase. Most of them are near the interface of subunits I and II. Several waters form a hydrogen-bonded cluster, including the heme propionates and the Mg͞Mn binding site. The F v fragment binds to the periplasmic polar domain of subunit II and is critically involved in the formation of the crystal lattice. The crystallization procedure is well reproducible and will allow for the analysis of the structures of mechanistically interesting mutant cytochrome c oxidases.

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confidence: 99%

Structure at 2.7 Å resolution of the Paracoccus denitrificans two-subunit cytochrome c oxidase complexed with an antibody F _V fragment

Ostermeier

Harrenga

Ermler

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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742

792

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“…Expression of Fv fragments and their secretion into the periplasmatic cell compartment in E. coli was achieved using the expression vector pASK68 (Kleymann et al, 1995b). The high expression level and the presence of the Strep-tag in the V, chain allowed us to use the recombinant Fv fragment for the one-step purification of the trimeric PSI complex by immunocomplex chromatography (Kleymann et al, 1995b). The extremely mild conditions employed for the elution permitted the isolation of the native PSI/Fv complex which exhibited full photoreactivity.…”

Section: Discussionmentioning

confidence: 99%

“…The high density of immunolabeled PSI reflects the relatively high amount of PSI present in thylakoid membranes (Melis, 1991). Expression of Fv fragments and their secretion into the periplasmatic cell compartment in E. coli was achieved using the expression vector pASK68 (Kleymann et al, 1995b). The high expression level and the presence of the Strep-tag in the V, chain allowed us to use the recombinant Fv fragment for the one-step purification of the trimeric PSI complex by immunocomplex chromatography (Kleymann et al, 1995b).…”

Section: Discussionmentioning

confidence: 99%

“…Total RNA was prepared from hybridoma cells (1 04-lo6 cells) using the guanidinium thiocyanate/cesium chloride procedure (Lanick et al, 1989) and employed for firststrand cDNA synthesis by oligo-dT priming, Amplification and cloning of the V, and V, gene domains were performed according to the method of Kleymann et al (1995b). First, the purified V, cDNA was cloned into the pASK68 plasmid.…”

Section: Methodsmentioning

confidence: 99%

“…For example, the streptavidin affinity tag (Strep-tag) of recombinant Fv fragments allows purification by affinity chromatography on streptavidin-Sepharose (Schmidt and Skerra, 1993). The bifunctionality of engineered Fv fragments in binding both the antigen and streptavidin makes a one-step purification of membrane proteins by immune-complex chromatography possible (Kleymann et al, 1995b).…”

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confidence: 99%

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Isolation and Structural Characterization of Trimeric Cyanobacterial Photosystem I Complex with the Help of Recombinant Antibody Fragments

Tsiotis

Haase

Engel

et al. 1995

European Journal of Biochemistry

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A monoclonal antibody was derived from mice immunized with the native trimeric, photosystem I (PSI) complex from the cyanobacterium Synechococcus PCC 7002 which reacts with a conformational epitope of the PSI complex. As seen by immunoelectron microscopy, the mAb bound to the stromal side of the thylakoid membranes. The DNA sequence encoding variable regions of the mAb was cloned into recombinant plasmids, sequenced and expressed in Escherichia coli. ELISA, Western blots and immunoelectron microscopy provided evidence that the expressed paired variable domain (Fv) fragments bind to the antigen in the same way as the parent mAb.A one-step purification was applied to purify the trimeric PSI complex using an affinity tag attached to the Fv fragment. Analysis by gel electrophoresis and N-terminal sequencing revealed the presence of the p s d , psaB, psaC, psaD, psaE, psaF and psaL gene products. The antenna size of the isolated PSI/ Fv was 139 k 9 chlorophyll a/primary electron donor. Flash-induced absorption-change measurements showed that the complex exhibited electron transfer from the primary electron donor, P700, to the Fe-S center, FAIF,. The position of the bound Fv fragment on the trimeric PSI surface was determined by highresolution electron microscopy and digital image processing.Keywords. Monoclonal antibody ; recombinant antibody ; immunoaffinity chromatography ; immuno-gold labeling ; high-resolution electron microscopy.Photosystem I (PSI) is a pigment-protein complex of the thylakoid membrane which mediates light-driven electron transfer from plastocyanin or cytochrome c553 to ferredoxin. The PSI complex in cyanobacteria and higher plants is comprised of at least 12 polypeptides, which are products of the psaA-psaL genes (see Goldbeek and Bryant, 1991, for review). The major component of the photoreaction center is a heterodimer composed of the psaAlpsaB gene products, each of which has a molecular mass of 80 kDa. The heterodimer carries the following components : the antenna chlorophylls, the primary electron donor P700, the primary electron acceptor &, an intermediate acceptor A, and the [4Fe-4S] cluster F, (Lagoute and Mathis, 1989). The molecular masses of the remaining protein subunits are 4-20 kDa. The subunits derived from the p s d , psal, psaK, psaL and psaM genes are hydrophobic and are predicted to possess trans-membrane a helices. Three of the four extrinsic subunits (the psaC, psaD and psaE gene products) are located on the stromal side, whereas the psaF subunit is located on the lumen side of the thylakoid membrane (Wynn and Malkin, 1988;Zanetti and Merati, 1987 Abbreviations. Chla, chlorophyll a ; C,,-M, dodecyl-P-D-maltoside ; Fv, paired variable domains; V,, heavy-chain variable region; V,, lightchain variable region; PSI, photosystem I; P, , , primary electron donor; STEM, scanning transmission electron microscopy.

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Bacterial CytochromecOxidase

Kannt

Michel

2004

Handbook of Metalloproteins

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Engineered Fv Fragments as a Tool for the One-Step Purification of Integral Multisubunit Membrane Protein Complexes

Cited by 95 publications

References 39 publications

Structure at 2.7 Å resolution of the Paracoccus denitrificans two-subunit cytochrome c oxidase complexed with an antibody F _V fragment

Structure at 2.7 Å resolution of the Paracoccus denitrificans two-subunit cytochrome c oxidase complexed with an antibody F _V fragment

Isolation and Structural Characterization of Trimeric Cyanobacterial Photosystem I Complex with the Help of Recombinant Antibody Fragments

Bacterial CytochromecOxidase

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Engineered Fv Fragments as a Tool for the One-Step Purification of Integral Multisubunit Membrane Protein Complexes

Cited by 95 publications

References 39 publications

Structure at 2.7 Å resolution of the Paracoccus denitrificans two-subunit cytochrome c oxidase complexed with an antibody F V fragment

Structure at 2.7 Å resolution of the Paracoccus denitrificans two-subunit cytochrome c oxidase complexed with an antibody F V fragment

Isolation and Structural Characterization of Trimeric Cyanobacterial Photosystem I Complex with the Help of Recombinant Antibody Fragments

Bacterial CytochromecOxidase

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Product

Resources

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Structure at 2.7 Å resolution of the Paracoccus denitrificans two-subunit cytochrome c oxidase complexed with an antibody F _V fragment

Structure at 2.7 Å resolution of the Paracoccus denitrificans two-subunit cytochrome c oxidase complexed with an antibody F _V fragment