Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus.

Janknecht, Ralf; Martynoff, Guy de; Lou, Jiatao; Hipskind, Robert A.; Nordheim, Alfred; Stunnenberg, Hendrik G.

doi:10.1073/pnas.88.20.8972

Cited by 429 publications

(241 citation statements)

References 17 publications

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“…The soluble fraction was separated by ultracentrifugation at 65,000 rpm, and the supernatant was applied to an Ni-nitrilotriacetic acid resin column and eluted according to the manufacturer (The QIAexpressionist; Janknecht et al, 1991); determination of protein concentration was performed by a Bradford assay (Bio-Rad).…”

Section: Purification Of Recombinant Myosin From T Gondiimentioning

confidence: 99%

A Dibasic Motif in the Tail of a Class XIV Apicomplexan Myosin Is an Essential Determinant of Plasma Membrane Localization

Hettmann

Herm

Geiter

et al. 2000

MBoC

135

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Obligate intracellular parasites of the phylum Apicomplexa exhibit gliding motility, a unique form of substrate-dependent locomotion essential for host cell invasion and shown to involve the parasite actin cytoskeleton and myosin motor(s). Toxoplasma gondii has been shown to express three class XIV myosins, TgM-A, -B, and -C. We identified an additional such myosin, TgM-D, and completed the sequences of a related Plasmodium falciparum myosin, PfM-A. Despite divergent structural features, TgM-A purified from parasites bound actin in an ATP-dependent manner. Isoform-specific antibodies revealed that TgM-A and recombinant mycTgM-A were localized right beneath the plasma membrane, and subcellular fractionation indicated a tight membrane association. Recombinant TgM-D also had a peripheral although not as sharply defined localization. Truncation of their respective tail domains abolished peripheral localization and tight membrane association. Conversely, fusion of the tails to green fluorescent protein (GFP) was sufficient to confer plasma membrane localization and sedimentability. The peripheral localization of TgM-A and of the GFP-tail fusion did not depend on an intact F-actin cytoskeleton, and the GFP chimera did not localize to the plasma membrane of HeLa cells. Finally, we showed that the specific localization determinants were in the very C terminus of the TgM-A tail, and site-directed mutagenesis revealed two essential arginine residues. We discuss the evidence for a proteinaceous plasma membrane receptor and the implications for the invasion process.

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Section: Purification Of Recombinant Myosin From T Gondiimentioning

confidence: 99%

A Dibasic Motif in the Tail of a Class XIV Apicomplexan Myosin Is an Essential Determinant of Plasma Membrane Localization

Hettmann

Herm

Geiter

et al. 2000

MBoC

135

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“…13 Five normal individuals were sensitized with 2% DCP (a nontoxic contact allergen used for the therapy of alopecia areata). One week later they were painted with a hydrophobic cream containing 1 mg/g of the purified TCR peptide or control peptide.…”

Section: Figure 4 Inhibitory Effects Of Ptcrtmb On Alloantigen-inducementioning

confidence: 99%

Therapeutic application of T cell receptor mimic peptides or cDNA in the treatment of T cell-mediated skin diseases

Göllner¹,

Müller²,

Alt³

et al. 2000

Gene Ther

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An 8-amino acid peptide encoding a sequence of the transmembrane region of the T cell receptor ␣ chain (TCR-␣) was shown to inhibit T cell function by preventing functional assembly of the T cell receptor (mimic peptide). To avoid systemic immunosuppression by peptide application in vivo, we used a topical application of the peptide. In the system of murine contact sensitivity, topical application of the peptide inhibited the elicitation of contact sensitivity following application of a contact allergen in sensitized animals. Alternatively, when naked DNA encoding the peptide sequence was injected into skin before application of a contact allergen to Keywords: TCR; T cell function; allergical contact dermatitis; T cell mediated disease; DNA vaccinationThe ␣, ␤ T cell receptor (TCR) is a multisubunit transmembrane complex composed of at least six different polypeptides (␣, ␤, ␥, ␦, ⑀ and ). 1 The TCR-␣ and -␤ subunits appear as a disulfide-linked homodimer that is the ligand binding site and determines the specificity of the receptor. For signal transduction into the cytoplasm, the TCR is noncovalently associated with the CD3 molecules (CD3-␥, -␦, -⑀ and -). The transmembrane region of the TCR is of critical importance for a functional assembly of the T cell receptor. 2 The transmembrane region of the TCR-␣ chain is highly conserved in different species. Studies on the assembly of the multisubunit TCR demonstrated that the stable interactions between the TCR-␣ chain and the CD3 molecule were located to eight amino acids within the transmembrane region of TCR-␣. [3][4][5] Manolios et al 6 demonstrated that an eight amino acid peptide mimicking the TCR-␣ chain inhibits T cell function in vitro and in vivo. These investigators showed the inhibitory effect of this peptide in different murine autoimmune disease models. The disadvantage of the systemic application of the mimic peptide is the general and long-lasting immunosuppressive effect that is unwanted for human applications.Allergic contact dermatitis (ACD) is an example for a T cell mediated delayed-type hypersensitivity (DTH) reaction. 7 ACD contains two main phases. During the sensitization phase epidermal Langerhans cells (LC) and

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“…Recombinant virus was generated and isolated by standard techniques and used to infect Spodoptera frugiperda cells (Sf9). For recombinant expression Sf9 cells were grown in FCS-free Express medium (BioWhittaker) and the recombinant proteins were purified by Ni2--NTA agarose chromatography [30,31], 120 h after infection. Purity of the recombinant fragments was examined by SDS-PAGE and found to be > 95%.…”

Section: Recombinant H Jragmentsmentioning

confidence: 99%

Analysis of the recognition mechanism of the alternative pathway of complement by monoclonal anti‐factor H antibodies: evidence for multiple interactions between H and surface bound C3b

et al. 1996

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The ability of the alternative pathway of complement to discriminate targets as either activators or non-activators is mediated by different binding properties of factor H to surfaceassociated C3b molecules. In the present study we have probed the interaction between H and C3b using five anti-H mAb. The binding sites of the mAb were mapped by Western blotting using both recombinant and trypsin-generated H fragments. Two mAb bound to CCP1 (90X, 196X), two to CCP5 (MRC OX24, 86X) and one to CCP8-15a (13IX). At a molar ratio 2:1 of 12si-ll :mAb all tested mAb enhanced binding of H to both activatorand non-activator-bound C3b. At higher concentrations two mAb had an inhibitory effect on H binding to surface-associated C3b (OX24, 131X). Thus the mAb 131X inhibits H binding to surface-bound C3b but unlike OX24 it does not bind to the previously described C3b binding site within or near CCP4-5. These results indicate that there is an additional interaction site on factor H for surface-bound C3b.

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Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus.

Cited by 429 publications

References 17 publications

A Dibasic Motif in the Tail of a Class XIV Apicomplexan Myosin Is an Essential Determinant of Plasma Membrane Localization

A Dibasic Motif in the Tail of a Class XIV Apicomplexan Myosin Is an Essential Determinant of Plasma Membrane Localization

Therapeutic application of T cell receptor mimic peptides or cDNA in the treatment of T cell-mediated skin diseases

Analysis of the recognition mechanism of the alternative pathway of complement by monoclonal anti‐factor H antibodies: evidence for multiple interactions between H and surface bound C3b

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