DE19908752A1 - New synthetic peptides from the Vpr protein of human immune deficiency virus, useful e.g. for therapy and diagnosis, have good solubility in water - Google Patents
New synthetic peptides from the Vpr protein of human immune deficiency virus, useful e.g. for therapy and diagnosis, have good solubility in waterInfo
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- DE19908752A1 DE19908752A1 DE19908752A DE19908752A DE19908752A1 DE 19908752 A1 DE19908752 A1 DE 19908752A1 DE 19908752 A DE19908752 A DE 19908752A DE 19908752 A DE19908752 A DE 19908752A DE 19908752 A1 DE19908752 A1 DE 19908752A1
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- arg
- glu
- leu
- ile
- gln
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
- C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Abstract
Description
Die Erfindung betrifft synthetische (s) Peptide des regulatorischen Virusproteins R (Vpr) des Humanen Immundefizienzvirus Typ 1 (HIV-1), insbesondere die chemische Totalsynthese des 96 Aminosäure langen Vpr-Proteins (sVpr1-96) sowie seiner Sequenzen. Als synthetische Vpr-Peptide finden sie Verwendung in biologischen Assays, in der Analyse der molekularen Struktur und den physikochemischen Eigenschaften von Vpr und dessen Domänen sowie zur Erzeugung von Antikörpern gegen Vpr-Peptidsequenzen.The invention relates to synthetic peptides of the regulatory virus protein R (Vpr) des Human immunodeficiency virus type 1 (HIV-1), in particular the chemical total synthesis of the 96 amino acid long Vpr protein (sVpr1-96) and its sequences. As synthetic Vpr peptides are used in biological assays, in molecular analysis Structure and physicochemical properties of Vpr and its domains as well as Generation of antibodies to Vpr peptide sequences.
Die bislang einzige in vitro charakterisierte biochemische Aktivität von Vpr ist die eines Kationen-selektiven Ionenkanals (Piller et al., 1996, - Literaturverzeichnis am Ende der Ausführungsbeispiele). Diese Arbeiten basierten auf der Annahme, daß die C-terminale alpha Helix (Positionen 46 bis 71 in Vpr), welche Ähnlichkeiten zu der Bienengift-Komponente Melittin besitzt, als Transmembrananker eine Membranpore ausbilden kann. Tatsächlich konnte rekombinantes, in Escherichia (E.) coli exprimiertes Vpr in künstlichen planaren Lipidbilayern rekonstituiert werden. Dadurch wurde eine durch das Membranpotential regulierbare Ionenkanalaktivität ermittelt, deren Regulierbarkeit von der basischen C terminalen Region abhängt, welche mit der negativ geladenen zytoplasmatischen Seite der Zellmembran in Wechselwirkung treten soll.The only biochemical activity of Vpr characterized in vitro so far is one Cation-selective ion channel (Piller et al., 1996, - bibliography at the end of the Embodiments). This work was based on the assumption that the C-terminal alpha Helix (positions 46 to 71 in Vpr), which are similar to the bee venom component Melittin has, as a transmembrane anchor, can form a membrane pore. Indeed was able to recombinant Vpr expressed in Escherichia (E.) coli in artificial planar Lipid bilayers are reconstituted. This made one through the membrane potential adjustable ion channel activity determined, the controllability of the basic C terminal region, which corresponds to the negatively charged cytoplasmic side of the Cell membrane should interact.
Es liegen Hinweise für Homooligomerisierung von Vpr vor: Ein rekombinantes Vpr- Fusionsprotein bildet oligomere Strukturen mit Molekulargewichten von <100 kDa (Zhao et al., 1994b), eine Beobachtung, die bislang an viralen Vpr nicht bestätigt wurde.There are indications of homooligomerization of Vpr: A recombinant Vpr Fusion protein forms oligomeric structures with molecular weights of <100 kDa (Zhao et al., 1994b), an observation that has not yet been confirmed on viral Vpr.
Untersuchungen zur molekularen Struktur von Vpr wurden durch zwei Gruppen mittels Sekundärstruktur-Analysen an kurzen Vpr-Peptiden durchgeführt: NMR-Studien an überlappenden Peptiden in wässerigem Trifluorethanol (TFE) sowie in Natriumdodecylsulfat(SDS)-Mizellen identifizierten alpha-helikale Regionen in den Vpr- Positionen 50-82. (Yao et al., 1998). Das Potential zur Helix-Bildung in der C-terminalen als auch der N-terminalen Region von Vpr wurde zuvor von verschiedenen Autoren vorhergesagt (Mahalingam et al., 1995a-d; Yao et al, 1995; Wang et al., 1996b). Neuere Studien mittels CD-Spektroskopie in TFE-haltigen Lösungen an 25 Aminosäure langen Peptiden (Luo et al., 1998) zeigten erste experimentelle Hinweise für die Existenz der N- und C-terminalen Helices in Vpr. Zahlreiche und zum Teil in ihrer Aussage kontroverse Mutationsanalysen haben versucht, die verschiedenen Primär- und Sekundärstrukturen einzelnen biologischen Aktivitäten von Vpr zuzuordnen (Mahalingam et al., 1995a-d, 1997; Wang et al., 1996a,b; Nie et al., 1998; Di Marzio et al., 1995).Studies on the molecular structure of Vpr were carried out by two groups Secondary structure analyzes performed on short Vpr peptides: NMR studies on overlapping peptides in aqueous trifluoroethanol (TFE) and in Sodium dodecyl sulfate (SDS) micelles identified alpha-helical regions in the Vpr Positions 50-82. (Yao et al., 1998). The potential for helix formation in the C-terminal as the N-terminal region of Vpr has also been predicted by various authors (Mahalingam et al., 1995a-d; Yao et al, 1995; Wang et al., 1996b). Recent studies using CD spectroscopy in TFE-containing solutions on 25 amino acid long peptides (Luo et al., 1998) showed the first experimental evidence for the existence of the N- and C-terminal helices in vpr. Have numerous and partly controversial mutation analyzes tries to biological the different primary and secondary structures Assign activities of Vpr (Mahalingam et al., 1995a-d, 1997; Wang et al., 1996a, b; Nie et al., 1998; Di Marzio et al., 1995).
Über die chemische Vollsynthese eines Vpr-Proteins wurde erstmals 1997 von Rocquigny und Mitarbeitern berichtet. Die Autoren beschrieben die Synthese eines 96 Aminosäure großen Peptides, welches von dem Virusisolat HIV-189,6 (Collman et al. 1992) abstammt. Neben den in dieser Arbeit beschriebenen Nachteilen (siehe im weiteren Text) ist dieses Protein in 9 Aminosäurepositionen unterschiedlich zu Vpr von HIV-1NL4-3, dessen Darstellung in der vorliegenden Erfindungsbeschreibung erstmalig berichtet wird. Somit besteht eine 10%-ige Divergenz zwischen den bereits beschriebenen (Rocquigny et al., 1997) und dem in den vorliegenden Verfahren dargestellten Produkten, welche die Gesamt- und Teilsequenzen des Vpr-Proteins von HIV-1NL4-3 (Adachi et al, 1986) betreffen.Rocquigny and co-workers first reported the full chemical synthesis of a Vpr protein in 1997. The authors described the synthesis of a 96 amino acid peptide derived from the virus isolate HIV-1 89.6 (Collman et al. 1992). In addition to the disadvantages described in this work (see further text), this protein is different in 9 amino acid positions from Vpr of HIV-1 NL4-3 , the presentation of which is reported for the first time in the present description of the invention. There is thus a 10% divergence between the previously described (Rocquigny et al., 1997) and the products presented in the present process, which show the total and partial sequences of the Vpr protein of HIV-1 NL4-3 (Adachi et al , 1986) concern.
Rocquigny und Mitarbeitern (1997) geben keine Angaben über die Reinheit sowie die physikochemischen Eigenschaften des Vpr-Peptides an. Es wird lediglich mittels der Far- Westernblot Technik gezeigt, daß SDS-denaturiertes Vpr-Peptid mit dem viralen Nukleoprotein NCp7 des gleichen HIV-1-Isolates in Wechselwirkung tritt. Dieser Befund der NCp7-Vpr-Wechselwirkung konnte bislang von keiner der zahlreichen anderen auf dem Vpr- Gebiet forschenden Gruppen bestätigt werden. Wesentlicher Nachteil dieser Vpr-Synthese ist die Tatsache, daß für dieses Peptid keine der beschriebenen biologischen Aktivitäten durch die Autoren gezeigt wurde. Insbesondere wird gezeigt, daß dieses Vpr-Peptid nicht an p6Gag bindet, eine weithin akzeptierte Eigenschaft von Vpr (Paxton et al., 1993; Lavallee et al, 1994; Kondo et al., 1995; Lu et al., 1995; Kondo und Göttlinger, 1996). Darüber hinaus wird beschrieben, daß dieses Peptid kerne Oligomeren bildet, und es liegen Hinweise vor, daß dieses Peptid in rein wässerigem System unlöslich ist. Von dem gleichen Labor wird in einer weiteren Studie (Roques et al., 1997) ein Modell der Vpr-NCp7-Wechselwirkung vorgestellt, welches auf Strukturanalysen an Teilsequenzen dieser Peptide basiert. Die Daten dazu werden jedoch in dieser Arbeit oder anderen Veröffentlichungen der Autoren nicht näher beschrieben.Rocquigny and co-workers (1997) do not give any information about the purity or the physicochemical properties of the Vpr peptide. It is only shown by means of the Far Western blot technique that SDS-denatured Vpr peptide interacts with the viral nucleoprotein NCp7 of the same HIV-1 isolate. This finding of the NCp7-Vpr interaction has so far not been confirmed by any of the numerous other groups researching in the Vpr field. A major disadvantage of this Vpr synthesis is the fact that none of the biological activities described has been shown by the authors for this peptide. In particular, it is shown that this Vpr peptide does not bind to p6 Gag , a widely accepted property of Vpr (Paxton et al., 1993; Lavallee et al, 1994; Kondo et al., 1995; Lu et al., 1995; Kondo and Göttlinger, 1996). In addition, it is described that this peptide forms nucleic oligomers and there are indications that this peptide is insoluble in a purely aqueous system. In a further study (Roques et al., 1997) from the same laboratory, a model of the Vpr-NCp7 interaction is presented, which is based on structural analyzes of partial sequences of these peptides. However, the data are not described in this work or other publications by the authors.
Teilsequenzen von Vpr (Positionen 50-75, 50-82 und 59-86) wurden für NMR-Studien an synthetischen Peptiden eingesetzt (Yao et al., 1998). Eine andere Gruppe hat zwei 25 Aminosäure lange Peptide aus den Bereichen der vorhergesagten alpha-helikalen Domänen in Vpr mittels CD-Spektroskopie untersucht (Luo et al., 1998):Partial sequences from Vpr (positions 50-75, 50-82 and 59-86) were used for NMR studies synthetic peptides (Yao et al., 1998). Another group has two 25 Amino acid long peptides from the areas of the predicted alpha-helical domains investigated in Vpr using CD spectroscopy (Luo et al., 1998):
Kurze, ca. 20 Aminosäure lange Peptide der C-terminalen Region von Vpr; welche das Motiv "HF/SRIG" enthalten, haben in einer Konzentration von 0.7 bis 3 micro-M zytotoxische Wirkungen gegenüber verschiedenen Hefe-Stämmen, wie zum Beispiel Saccharomyces cerevisiae, Candida albicans und Schizosaccharomyces pombe (Macreadie et al., 1996, 1997) auslöst. Eine erhöhte Konzentration von bivalenten Kationen, insbesondere Magnesium und Kalzium, verhindert die Aufnahme der Vpr-Peptide und dadurch deren toxische Effekte. Weiterführende Studien zeigten, daß ein C-terminales Vpr-Peptid (Positionen 71-82) die Membranpermeabilisierung, weiterhin eine Reduktion des Mitochondrienmembranpotentials und letztendlich den Zelltod von CD4+ T-Zellen bewirkt (Macreadie et al., 1997). Schließlich wurden ähnliche toxische Effekte ebenfalls für Gesamt-Vpr demonstriert (Arunagiri ef al., 1997). Dazu wurde das gleiche rekombinante Glutathione S-Transferase(GST)-Vpr- Fusionsprotein eingesetzt, welches zuvor für Ionenkanalstudien an Vpr verwendet wurde (Piller et al., 1996). Jedoch berichten die Autoren ebenfalls über Probleme mit der Löslichkeit des rekombinanten Produktes in wässerigen Systemen.Short, about 20 amino acid long peptides of the C-terminal region of Vpr; which contain the motif "HF / SRIG" have a concentration of 0.7 to 3 micro-M cytotoxic effects against various yeast strains, such as Saccharomyces cerevisiae, Candida albicans and Schizosaccharomyces pombe (Macreadie et al., 1996, 1997) triggers. An increased concentration of divalent cations, especially magnesium and calcium, prevents the uptake of the Vpr peptides and thereby their toxic effects. Further studies showed that a C-terminal Vpr peptide (positions 71-82) causes membrane permeabilization, a further reduction in the mitochondrial membrane potential and ultimately cell death of CD4 + T cells (Macreadie et al., 1997). Finally, similar toxic effects were also demonstrated for total Vpr (Arunagiri ef al., 1997). The same recombinant glutathione S-transferase (GST) -Vpr fusion protein was used, which was previously used for ion channel studies on Vpr (Piller et al., 1996). However, the authors also report problems with the solubility of the recombinant product in aqueous systems.
Rekombinantes Vpr des Isolates HIV-1NL4-3 wurde in Insektenzellen nach Infektion mit
rekombinanten Baculoviren exprimiert (Levy et all, 1995). Die Reinigung des Produktes
erfolgte lediglich durch Immunaffilnitätschromatographie an immobilisiertem polyklonalen
Antiserum, welches gegen die N-terminale Domäne von Vpr gerichtet ist. Dazu wurden
Zellkulturüberstände eingesetzt, da rekombinantes Vpr unspezifisch in das Kulturmedium
sekretiert wird. Reinigungsstrategien für die Produktion größerer Mengen an rekombinanten
Vpr wurden nicht beschrieben. In den meisten Fällen wurden von Autoren Vpr-haltige
Zellkulturüberstände für biologische Tests verwendet. Dabei konnte gezeigt werden, daß
rekombinantes Vpr die Virusreplikation in PBMC (peripheral blood mononuclear cells) und in
verschiedenen latent infizierten Monozyten- und T-Zellinien aktiviert. Wesentliche Nachteile
dieses Verfahrens sind:
Recombinant Vpr of the isolate HIV-1 NL4-3 was expressed in insect cells after infection with recombinant baculoviruses (Levy et all, 1995). The purification of the product was carried out only by immunoaffinity chromatography on immobilized polyclonal antiserum which is directed against the N-terminal domain of Vpr. Cell culture supernatants were used for this, since recombinant Vpr is secreted non-specifically into the culture medium. Purification strategies for the production of large amounts of recombinant Vpr have not been described. In most cases, authors used Vpr-containing cell culture supernatants for biological tests. It was shown that recombinant Vpr activates virus replication in PBMC (peripheral blood mononuclear cells) and in various latently infected monocyte and T cell lines. The main disadvantages of this procedure are:
- - geringe Ausbeute und keine Möglichkeit zur Herstellung von mg-Mengen an hochreinem Produkt;- Low yield and no possibility of producing mg amounts of high purity Product;
- - rekombinantes Vpr wurde im Prozeß der Affinitätsreinigung mit Detergentien versetzt, wodurch Dialyse und Renaturierung notwendig wurden;- recombinant Vpr was mixed with detergents in the process of affinity purification, which made dialysis and renaturation necessary;
- - Studien zu einer möglichen posttranslationalen Modifizierung von Vpr in Insektenzellen wurden nicht beschrieben;- Studies on a possible post-translational modification of Vpr in insect cells were not described;
- - die Wirkung von rekombinanten Vpr in HIV-infizierten primären Monozyten/Makrophagen wurde nicht getestet.- the effect of recombinant Vpr in HIV-infected primary monocytes / macrophages has not been tested.
Expression, Reinigung sowie biochemische Charakterisierung von rekombinanten Vpr wurden erstmals 1994 von Zhao und Mitarbeitern beschrieben. Dazu wurde die kodierende Sequenz des Vpr-Proteins des Isolates HIV-189,6 in E. coli als Fusionsprotein exprimiert. Zum Zweck der Reinigung und des Nachweises wurde in diesem Verfahren C-terminal eine 25 Aminosäuren lange Sequenz des heterologen FLAG-Epitopes fusioniert. Außer der Oligomerisierung wurde über keine biologischen Aktivitäten des rekombinanten Produktes in dieser Arbeit berichtet. Wesentlicher Nachteil dieses Verfahrens ist die Tatsache, daß Vpr nicht in seiner authentischen Sequenz, sondern als Fusionsprotein exprimiert wird.Expression, purification and biochemical characterization of recombinant Vpr were first described in 1994 by Zhao and co-workers. For this purpose, the coding sequence of the Vpr protein of the isolate HIV-1 89.6 was expressed in E. coli as a fusion protein. For the purpose of purification and detection, a 25 amino acid sequence of the heterologous FLAG epitope was fused C-terminally in this method. Apart from oligomerization, no biological activities of the recombinant product were reported in this work. A major disadvantage of this method is the fact that Vpr is not expressed in its authentic sequence, but as a fusion protein.
In einem weiteren Verfahren wurde Vpr des Isolates HIV-1HXB2 in E. coli als GST- Fusionsprotein exprimiert (Piller et al., 1996). Nach Affinitätschromatographie an Glutathione- Agarose wurde Vpr durch Thrombin-Spaltung vom Fusionsanteil befreit. Wesentlicher Nachteil dieses Verfahrens ist die Tatsache, daß Vpr nach Spaltung eine starke Tendenz zur Aggregation besitzt und nicht in wässeriger Lösung gehalten werden kann. So berichten zum Beispiel Arunagiri und Mitarbeiter (1997), daß mit diesem Verfahren hergestelltes rekombinantes Vpr nach Abspaltung des GST-Fusionsanteils nicht in Lösung gehalten werden kann, sondern nur durch Beibehaltung des heterologen Fusionsanteils Vpr in wässerigen Systemen getestet werden konnte.In a further method, Vpr of the isolate HIV-1 HXB2 was expressed in E. coli as a GST fusion protein (Piller et al., 1996). After affinity chromatography on glutathione agarose, Vpr was freed of the fusion portion by thrombin cleavage. A major disadvantage of this method is the fact that Vpr has a strong tendency to aggregate after cleavage and cannot be kept in aqueous solution. For example, Arunagiri and co-workers (1997) report that recombinant Vpr produced with this method cannot be kept in solution after the GST fusion fraction has been split off, but can only be tested in aqueous systems by maintaining the heterologous fusion fraction Vpr.
In der Patentanmeldung WO 95/26361 (Azad, A.A., Macreadie, I.G., Arunagiri, C., 1995) werden biologisch aktive Peptidfragmente des Vpr-Proteins von HIV beschrieben; pharmazeutische Verbindungen, welche diese Peptide oder biologisch aktive Analoga davon enthalten; Antagonisten der Vpr-Peptide sowie pharmazeutische Verbindungen, welche diese Vpr-Antagonisten enthalten. Die chemische Synthese von Gesamt-Vpr-Protein spielt darin keine Rolle.In patent application WO 95/26361 (Azad, A.A., Macreadie, I.G., Arunagiri, C., 1995) biologically active peptide fragments of the Vpr protein from HIV are described; pharmaceutical compounds containing these peptides or biologically active analogues thereof contain; Antagonists of the Vpr peptides as well as pharmaceutical compounds, which these Vpr antagonists included. The chemical synthesis of total Vpr protein plays a role in this not matter.
In der WO 96/07741 (Cohen, E.; Bergeron, D.; Checroune, F.; Yao, X.-J:; Pignac-Kobinger, G., 1996) werden chimere Moleküle unter Schutz gestellt, bestehend aus Vpr von HIV-1 und Vpx von HIV-2, welche spezifisch in HIV-1/HIV-2-Viruspartikel eingebaut werden können und dort die strukturelle Organisation und funktionelle Integrität von Virionen stören. Sie sind jedoch für den Einsatz zur Gentherapie von HIV-1/HIV-2-Infektionen ausgeschlossen.In WO 96/07741 (Cohen, E .; Bergeron, D .; Checroune, F .; Yao, X.-J :; Pignac-Kobinger, G., 1996) protect chimeric molecules consisting of Vpr from HIV-1 and Vpx of HIV-2, which can be specifically incorporated into HIV-1 / HIV-2 virus particles and there disrupt the structural organization and functional integrity of virions. they are however excluded for use in gene therapy for HIV-1 / HIV-2 infections.
In WO 96/08970 (Weiner, D.B.; Levy, D.N.; Refaeli, Y., 1996) werden Methoden zur Inhibierung der Zellteilung und der Lymphozyten-Aktivierung unter Anwendung von Vpr- Proteinen, Fragmenten von Vpr oder Gensequenzen von Vpr beschrieben. Die chemische Synthese von Vpr-Proteinen spielt darin keine Rolle.WO 96/08970 (Weiner, D.B .; Levy, D.N .; Refaeli, Y., 1996) describes methods for Inhibition of cell division and lymphocyte activation using Vpr- Proteins, fragments of Vpr or gene sequences of Vpr are described. The chemical Synthesis of Vpr proteins plays no role in this.
Die Verwendung von vpr Genen im screening assay für anti-HIV-Arzneimittel wird in den US- Patenten 5721104 und 5639619 beschrieben, zur Bestimmung von HIV-2 in US 5580739, ein Vpr-Rezeptor-Protein in US 5780238.The use of vpr genes in the screening assay for anti-HIV drugs is described in the U.S. Patents 5721104 and 5639619 for determining HIV-2 in US 5580739 Vpr receptor protein in US 5780238.
Der Erfindung liegt die Aufgabe zugrunde, einen Syntheseweg für Vpr-Peptide im mg- Maßstab zu entwickeln, ihre Reinigung zu ermöglichen, und der Allgemeinheit das Endprodukt zur Verfügung zu stellen.The invention has for its object to provide a synthetic route for Vpr peptides in mg Develop scale, enable their cleaning, and the general public the final product to provide.
Die Aufgabe wurde erfindungsgemäß durch die Bereitstellung des Proteins sVpr1-96 sowie
der Peptide
The object was achieved according to the invention by the provision of the protein sVpr1-96 and the peptides
- - ein 47 Aminosäuren langes N-terminales Peptid (sVpr1-47),A 47 amino acid long N-terminal peptide (sVpr1-47),
- - ein 49 Aminosäuren langes C-terminales Peptid (sVpr48-96) und von Fragmenten dieser Peptide, zum Beispiel- A 49 amino acid long C-terminal peptide (sVpr48-96) and fragments thereof Peptides, for example
- - überlappende, etwa 15 Aminosäuren lange Peptide für die Epitop-Charakterisierung und isolelektrische Fokussierung- Overlapping, about 15 amino acid long peptides for epitope characterization and isolelectric focusing
-
- etwa 20 Aminosäuren lange Peptide zur strukturellen und funktionellen Charakterisierung
einzelner Domänen von Vpr, insbesondere die Peptide sVpr1-20 und sVpr21-40 gelöst:
sVpr1-96:
H-Met-Glu-Gln-Ala-Pro-Glu-Asp-Gln-Gly-Pro-Gln-Arg-Glu-Pro-Tyr-Asn-Glu-Trp-Thr-Leu-Glu- Leu-Leu-Glu-Glu-Leu-Lys-Ser-Glu-Ala-Val-Arg-His-Phe-Pro-Arg-Ile-Trp-Leu-His-Asn-Leu-Gly- Gln-His-Ile-Tyr-Glu-Thr-Tyr-Gly-Asp-Thr-Trp-Ala-Gly-Val-Glu-Ala-Ile-Ile-Arg-Ile-Leu-Gln-Gln- Leu-leu-Phe-Ile-His-Phe-Arg-Ile-Gly-Cys-Arg-His-Ser-Arg-Ile-Gly-Val-Thr-Arg-Gln-Arg-Arg-Ala- Arg-Asn-Gly-Ala-Ser-Arg-Ser-OH
sVpr1-47:
H-Met-Glu-Gln-Ala-Pro-Glu-Asp-Gln-Gly-Pro-Gln-Arg-Glu-Pro-Tyr-Asn-Glu-Trp-Thr-Leu-Glu- Leu-Leu-Glu-Glu-Leu-Lys-Ser-Glu-Ala-Val-Arg-His-Phe-Pro-Arg-Ile-Trp-Leu-His-Asn-Leu-Gly- Gln-His-Ile-Tyr-NH2;
sVpr48-96:
Glu-Thr-Tyr-Gly-Asp-Thr-Trp-Ala-Gly-Val-Glu-Ala-Ile-Ile-Arg-Ile-Leu-Gln-Gln-Leu-leu-Phe-Ile- His-Phe-Arg-Ile-Gly-Cys-Arg-His-Ser-Arg-Ile-Gly-Val-Thr-Arg-Gln-Arg-Arg-Ala-Arg-Asn-Gly- Ala-Ser-Arg-Ser-OH
sVpr1-20 als sVpr1-20(Asn5,10,14):
H-Met-Glu-Gln-Ala-Asn-Glu-Asp-Gln-Gly-Asn-Gln-Arg-Glu-Asn-Tyr-Asn-Glu-Trp-Thr-Leu-NH2 und
sVpr21-40 als sVpr2 l-40(Asn35):
H-Glu-Leu-Leu-Glu-Glu-Leu-Lys-Ser-Glu-Ala-Val-Arg-His-Phe-Asn-Arg-Ile-Trp-Leu-His-NH2, Fragmente dieser Peptide - mit etwa 15 Aminosäuren langen Peptiden
sVpr11-25:
Gln-Arg-Glu-Pro-Tyr-Asn-Glu-Trp-Thr-Leu-Glu-Leu-Leu-Glu-Glu-,
sVpr41-55:
Asn-Leu-Gly-Gln-His-Ile-Tyr-Glu-Thr-Tyr-Gly-Asp-Thr-Trp-Ala,
sVpr46-60:
Ile-Tyr-Glu-Thr-Tyr-Gly-Asp-Thr-Trp-Ala-Gly-Val-Glu-Ala-Ile-,
sVpr56-70:
Gly-Val-Glu-Ala-Ile-Ile-Arg-Ile-Leu-Gln-Gln-Leu-leu-Phe-Ile,
sVpr66-80:
Gln-Leu-leu-Phe-Ile-His-Phe-Arg-Ile-Gly-Cys-Arg-His-Ser-Arg,
sVpr76-96:
Cys-Arg-His-Ser-Arg-Ile-Gly-Val-Thr-Arg-Gln-Arg-Arg-Ala-Arg-Asn-Gly-Ala-Ser-Arg-Ser-OH.- About 20 amino acid long peptides for the structural and functional characterization of individual domains of Vpr, especially the peptides sVpr1-20 and sVpr21-40 solved:
sVpr1-96:
H-Met-Glu-Gln-Ala-Pro-Glu-Asp-Gln-Gly-Pro-Gln-Arg-Gl-Pro-Tyr-Asn-Glu-Trp-Thr-Leu-Glu- Leu-Leu-Glu- Glu-Leu-Lys-Ser-Glu-Ala-Val-Arg-His-Phe-Pro-Arg-Ile-Trp-Leu-His-Asn-Leu-Gly- Gln-His-Ile-Tyr-Glu-Thr- Tyr-Gly-Asp-Thr-Trp-Ala-Gly-Val-Glu-Ala-Ile-Ile-Arg-Ile-Leu-Gln-Gln- Leu-leu-Phe-Ile-His-Phe-Arg-Ile- Gly-Cys-Arg-His-Ser-Arg-Ile-Gly-Val-Thr-Arg-Gln-Arg-Arg-Ala- Arg-Asn-Gly-Ala-Ser-Arg-Ser-OH
sVpr1-47:
H-Met-Glu-Gln-Ala-Pro-Glu-Asp-Gln-Gly-Pro-Gln-Arg-Gl-Pro-Tyr-Asn-Glu-Trp-Thr-Leu-Glu- Leu-Leu-Glu- Glu-Leu-Lys-Ser-Glu-Ala-Val-Arg-His-Phe-Pro-Arg-Ile-Trp-Leu-His-Asn-Leu-Gly-Gln-His-Ile-Tyr-NH 2 ;
sVpr48-96:
Glu-Thr-Tyr-Gly-Asp-Thr-Trp-Ala-Gly-Val-Glu-Ala-Ile-Ile-Arg-Ile-Leu-Gln-Gln-Leu-leu-Phe-Ile- His-Phe- Arg-Ile-Gly-Cys-Arg-His-Ser-Arg-Ile-Gly-Val-Thr-Arg-Gln-Arg-Arg-Ala-Arg-Asn-Gly-Ala-Ser-Arg-Ser-OH
sVpr1-20 as sVpr1-20 (Asn 5,10,14 ):
H-Met-Glu-Gln-Ala-Asn-Glu-Asp-Gln-Gly-Asn-Gln-Arg-Glu-Asn-Tyr-Asn-Glu-Trp-Thr-Leu-NH 2 and
sVpr21-40 as sVpr2 l-40 (Asn 35 ):
H-Glu-Leu-Leu-Glu-Glu-Leu-Lys-Ser-Glu-Ala-Val-Arg-His-Phe-Asn-Arg-Ile-Trp-Leu-His-NH 2 , fragments of these peptides - with peptides about 15 amino acids long
sVpr11-25:
Gln-Arg-Glu-Pro-Tyr-Asn-Glu-Trp-Thr-Leu-Glu-Leu-Leu-Glu-Glu-,
sVpr41-55:
Asn-Leu-Gly-Gln-His-Ile-Tyr-Glu-Thr-Tyr-Gly-Asp-Thr-Trp-Ala,
sVpr46-60:
Ile-Tyr-Glu-Thr-Tyr-Gly-Asp-Thr-Trp-Ala-Gly-Val-Glu-Ala-Ile-,
sVpr56-70:
Gly-Val-Glu-Ala-Ile-Ile-Arg-Ile-Leu-Gln-Gln-Leu-leu-Phe-Ile,
sVpr66-80:
Gln-Leu-leu-Phe-Ile-His-Phe-Arg-Ile-Gly-Cys-Arg-His-Ser-Arg,
sVpr76-96:
Cys-Arg-His-Ser-Arg-Ile-Gly-Val-Thr-Arg-Gln-Arg-Arg-Ala-Arg-Asn-Gly-Ala-Ser-Arg-Ser-OH.
Die Synthese der C-terminalen Vpr-Peptide erfolgte an einem Serin-Harz mit Hilfe eines Perkin-Elmer-Synthesizers. Alle N-terminalen Peptide wurden an einem Polystyren- Polyoxyethylen-Trägerharz synthetisiert. Der Aufbau der Peptide erfolgte mittels FMOC(Fluormethyloxycarbonyl)-Strategie unter Verwendung von Schutzgruppen. Nach Beendigung der Synthese erfolgte die Abspaltung der Schutzgruppen mittels eines Abspaltungsgemisches, bestehend aus 95% Trifluoressigsäure, der 3% Triisopropylsilan und je nach Peptid 2 bis 5% Ethandithiol zugesetzt wurde. Das Harz wurde abgetrennt, die Reaktionslösung eingeengt und mit Heptan versetzt. Es wurde erneut eingeengt und das verbleibende Öl mit Diethylether digeriert. Das rohe Peptid wurde abgesaugt und anschließend aus Essigsäure lyophilisiert. Zur Reinigung wurden die Rohpeptide an einer präparativen HPLC-Anlage (High Pressure Liquid Chromatography) chromatographiert. Alle Peptide wurden an einer Kieselgelsäule mittels eines linearen Gradienten, bestehend aus TFA (Trifluoressigsäure) in Wasser und TFA in Acetonitril gereinigt. Die Eluate wurden eingeengt und lyophilisiert.The C-terminal Vpr peptides were synthesized on a serine resin using a Perkin Elmer synthesizers. All N-terminal peptides were isolated on a polystyrene Synthetic polyoxyethylene resin. The peptides were constructed using FMOC (fluoromethyloxycarbonyl) strategy using protecting groups. To The synthesis was terminated by means of a protective group Cleavage mixture consisting of 95% trifluoroacetic acid, the 3% triisopropylsilane and depending on the peptide, 2 to 5% ethanedithiol was added. The resin was separated, the The reaction solution was concentrated and heptane was added. It was narrowed down again and that remaining oil digested with diethyl ether. The crude peptide was aspirated and then lyophilized from acetic acid. The crude peptides were purified on a preparative HPLC system (High Pressure Liquid Chromatography) chromatographed. All Peptides were grown on a silica gel column using a linear gradient consisting of TFA (Trifluoroacetic acid) in water and TFA in acetonitrile. The eluates were concentrated and lyophilized.
Überraschenderweise hat sich herausgestellt, daß die erfindungsgemäß hergestellten sVpr- Peptide nach dieser Reinigungsprozedur - im Unterschied zu den bislang beschriebenen rekombinanten oder synthetischen Produkten - wasserlöslich sind und selbst in hohen Konzentration von bis zu mM-Lösungen keiner Proteinaggregation unterliegen. Es konnte gezeigt werden, daß das Protein sVpr1-96 eine gefaltete Struktur annimmt, biologische Aktivitäten vergleichbar mit viralen Vpr hat und immunologisch reaktiv ist.Surprisingly, it has been found that the sVpr- Peptides after this cleaning procedure - in contrast to the previously described recombinant or synthetic products - are water soluble and even in high Concentration of up to mM solutions are not subject to protein aggregation. It could are shown that the protein sVpr1-96 takes a folded structure, biological Has activities comparable to viral Vpr and is immunologically reactive.
Erstmals wird die chemische Synthese des Vpr-Proteins und seiner Fragmente beschrieben, welcher der Aminosäuresequenz des Virusisolates HIV-1NL4-3 entspricht.For the first time the chemical synthesis of the Vpr protein and its fragments is described, which corresponds to the amino acid sequence of the virus isolate HIV-1 NL4-3 .
Unter dem Begriff synthetische (s) Vpr-Peptide werden im Rahmen der vorliegenden Erfindungsbeschreibung die durch Festphasensynthese hergestellten Peptide verstanden, welche die authentische Aminosäuresequenz des nativen Vpr-Proteins enthalten, so wie dieses durch das vpr Gen des molekularen Isolates HIV-1NL4-3 kodiert wird.In the context of the present description of the invention, the term synthetic (s) Vpr peptides are understood to mean the peptides produced by solid-phase synthesis which contain the authentic amino acid sequence of the native Vpr protein, such as this by the vpr gene of the molecular isolate HIV-1 NL4-3 is encoded.
Das Wesen der Erfindung liegt in einer Kombination bekannter Merkmale (Ausgangsstoffe, Syntheseharze, Synthesizer) und neuer Lösungswege - der erstmaligen chemischen Synthese dieser Verbindungen, der Synthesestrategie, der Wahl der spezifischen Schutzgruppen, dem erfindungsgemäßen Abspaltungsgemisch Trifluoressigsäure- Triisopropylsilan-Ethandithiol, dem Einsatz eines bestimmten Lösungsmittelgradienten (TFA- Wasser-: TFA-Acetonitril für die Reinigung - die sich gegenseitig beeinflussen und in ihrer neuen Gesamtwirkung einen Gebrauchsvorteil und den erstrebten Erfolg ergeben, der darin liegt, daß nunmehr neue synthetisch hergestellte sVpr-Peptide zur Verfügung stehen.The essence of the invention lies in a combination of known features (starting materials, Synthetic resins, synthesizers) and new solutions - the first chemical Synthesis of these compounds, the synthesis strategy, the choice of the specific Protecting groups, the trifluoroacetic acid cleavage mixture according to the invention Triisopropylsilane-ethanedithiol, the use of a certain solvent gradient (TFA- Water: TFA acetonitrile for cleaning - which influence each other and in their new overall effect give a benefit in use and the desired success, which is in it is that new synthetically produced sVpr peptides are now available.
Die erfindungsgemäß hergestellten synthetischen Peptide zeichnen sich durch folgende
Eigenschaften aus:
Sie haben eine extrem gute Löslichkeit in wässerigen Systemen, welche bis zu mM
konzentrierte Peptid-Lösungen erlauben. Dies wiederum ist Voraussetzung für nachfolgende
Strukturanalysen von Vpr mittels NMR(Nuclear Magnetic Resonance)-spektroskopischer und
RKSA(Röntgenkristallstrukturanalyse)-Techniken.The synthetic peptides produced according to the invention are distinguished by the following properties:
They have an extremely good solubility in aqueous systems which allow up to mM concentrated peptide solutions. This in turn is a prerequisite for subsequent structural analyzes of Vpr using NMR (Nuclear Magnetic Resonance) spectroscopic and RKSA (X-ray crystal structure analysis) techniques.
Die Peptide lassen sich unter ökonomisch vertretbaren Bedingungen im mg-Maßstab herstellen und bis zu einem hohen Reinheitsgrad anreichern. Sie zeigen immunogene und biologische Eigenschaften, welche identisch sind mit denen von natürlichen Vpr-Proteinen. Sie lassen sich für vielfältige Gebiete der Grundlagenforschung sowie der angewandten Forschung auf dem Gebiet der HIV-Virologie einsetzen. The peptides can be economically justified on a mg scale manufacture and enrich to a high degree of purity. They show immunogenic and biological properties that are identical to those of natural Vpr proteins. she can be used for diverse areas of basic research as well as applied Use research in the field of HIV virology.
Die erfindungsgemäßen Peptide finden Verwendung in biologischen Assays, in der Strukturanalyse von Vpr und dessen Domänen, zur Erzeugung von Antikörpern gegen HIV- Peptidsequenzen, in antiviralen Reagenzien, zum Aufbau von Testsystemen zum Screenen von potentiellen Vpr-Antagonisten, bei der Etablierung von Zellkultur- und Tiermodellen, zur Untersuchung der Pathomechanismen von Vpr, für die in vitro Assemblierung von neuartigen Vektoren für den Einsatz bei Gentransfermethoden in der Gentherapie und zur Entwicklung von serologischen Testmethoden, insbesondere eines Vpr-Antigen-ELISA.The peptides according to the invention are used in biological assays in which Structural analysis of Vpr and its domains, for the generation of antibodies against HIV Peptide sequences, in antiviral reagents, for the construction of test systems for screening of potential Vpr antagonists in the establishment of cell culture and animal models, for Investigation of the pathomechanisms of Vpr, for the in vitro assembly of novel Vectors for use in gene transfer methods in gene therapy and for development of serological test methods, in particular a Vpr antigen ELISA.
Die erfindungsgemäß hergestellten Produkte können für die Aufklärung der molekularen Struktur von Vpr mittels NMR- und CD-spektrokopischen Methoden sowie der Kristallisation und nachfolgender RKSA eingesetzt werden. Diese Informationen wiederum sind essentiell für das Verständnis der molekularen Wirkungsweise des Vpr-Proteins im HIV-1- Replikationszyklus und der damit verbundenen Pathomechanismen einer AIDS-Erkrankung sowie dem molekularen Design von potentiellen Vpr-Antagonisten.The products produced according to the invention can be used for the elucidation of the molecular Structure of Vpr using NMR and CD spectroscopic methods as well as crystallization and subsequent RKSA can be used. This information, in turn, is essential for understanding the molecular mode of action of the Vpr protein in HIV-1 Replication cycle and the associated pathomechanisms of an AIDS disease and the molecular design of potential Vpr antagonists.
Weiterhin können mit diesen Produkten in vitro Testsysteme dargestellt werden, welche das intensive Screening von potentiellen anti-Vpr-wirksamen Reagenzien erlauben. Darüber hinaus können sie für die Erzeugung und Testung von Vpr-spezifischen Antikörpern und für serologische Testverfahren angewendet werden.In addition, these products can be used to display in vitro test systems which do this allow intensive screening of potential anti-Vpr-active reagents. About that They can also be used for the generation and testing of Vpr-specific antibodies and for serological test procedures are used.
Die Erfindung wird in der Peptidchemie, der virologischen Grundlagenforschung, der Strukturanalyse sowie der medizinischen Diagnostik angewendet.The invention is used in peptide chemistry, basic virological research, Structural analysis and medical diagnostics applied.
Sie soll anhand von Ausführungsbeispielen näher erläutert werden, ohne auf sie beschränkt zu sein.It is to be explained in more detail using exemplary embodiments, without being limited to them to be.
Die Synthese der C-terminalen Vpr-Peptide erfolgte an einem Serin-Harz der Fa. Rapp Polymere Tübingen an einem ABI 433A Synthesizer (Perkin Elmer).The C-terminal Vpr peptides were synthesized on a serine resin from Rapp Polymer Tübingen on an ABI 433A synthesizer (Perkin Elmer).
Alle N-terminalen Peptide wurden an einem Polystyren-polyoxyethylen-Trägerharz (TentaGel R-RAM-Harz der Fa. Rapp Polymere) synthetisiert.All N-terminal peptides were carried out on a polystyrene-polyoxyethylene carrier resin (TentaGel R-RAM resin from Rapp Polymer).
Der Aufbau der Peptide erfolgte mittels FMOC(Fluormethyloxycarbonyl)-Strategie unter Verwendung nachfolgender Schutzgruppen: O-t.Butylester für Glu und Asp, OtBu-Ether für Serin, Tyrosin und Threonin, Boc (tert-Butoxycarbonyl-) für Lysin und Tryptophan, Trt (Trityl - Triphenylmethyl-) für Histidin, Glutamin und Asparagin sowie Pbf (2.2.4.6.7-pentamethyl dihydrobenzofuran-5-sulfonyl-) für Arginin.The peptides were built using the FMOC (fluoromethyloxycarbonyl) strategy Use of the following protective groups: O-t-butyl ester for Glu and Asp, OtBu ether for Serine, tyrosine and threonine, Boc (tert-butoxycarbonyl-) for lysine and tryptophan, Trt (trityl - Triphenylmethyl-) for histidine, glutamine and asparagine as well as Pbf (2.2.4.6.7-pentamethyl dihydrobenzofuran-5-sulfonyl-) for arginine.
Nach Beendigung der Synthese erfolgte die Abspaltung der Schutzgruppen mittels eines Abspaltungsgemisches, bestehend aus 95% Trifluoressigsäure, der 3% Triisopropylsilan und je nach Peptid 2 bis 5% Ethandithiol zugesetzt wurde. Das Harz wurde abgetrennt, die Reaktionslösung eingeengt und mit Heptan versetzt. Es wurde erneut eingeengt und das verbleibende Öl mit Diethylether digeriert. Das rohe Peptid wurde abgesaugt und anschließend aus 10%iger Essigsäure lyophilisiert.After the synthesis had ended, the protective groups were cleaved off using a Cleavage mixture consisting of 95% trifluoroacetic acid, the 3% triisopropylsilane and depending on the peptide, 2 to 5% ethanedithiol was added. The resin was separated, the The reaction solution was concentrated and heptane was added. It was narrowed down again and that remaining oil digested with diethyl ether. The crude peptide was aspirated and then lyophilized from 10% acetic acid.
Zur Reinigung wurden jeweils 100 mg Rohpeptid an einer präparativen HPLC-Anlage (Shimadzu LC-8 Anlage) chromatographiert. Alle Peptide wurden an einer Kieselgelsäule (300 × 400 mm Vydac-RP18-Säule, Korngröße 15-20 µM) mittels eines linearen Gradienten, bestehend aus A = 1% TFA (Trifluoressigsäure) in Wasser und B = 0,1% TFA in 80%igem Acetonitril mit einem Fluss von 100 ml/min gereinigt. Die Eluate wurden eingeengt und lyophilisiert.For purification, 100 mg of crude peptide were used on a preparative HPLC system (Shimadzu LC-8 system) chromatographed. All peptides were on a silica gel column (300 × 400 mm Vydac-RP18 column, particle size 15-20 µM) using a linear gradient, consisting of A = 1% TFA (trifluoroacetic acid) in water and B = 0.1% TFA in 80% Acetonitrile cleaned at a flow of 100 ml / min. The eluates were concentrated and lyophilized.
Das Peptid wurde an einem TentaGel S-AC-Harz (0,20 mmol/Gramm) an einem ABI 433
aufgebaut. Am Schluß der Synthese wurde die FMOC-Schutzgruppe abgespalten, das Harz
nacheinander mit Dimethylformamid und Methylenchlorid gewaschen und getrocknet. Das
Peptid wurde dann in der eingangs beschriebenen Weise vom Harz abgespalten und
anschließend gereinigt.
Molmasse: 11378 gef. 11381
H-Met-Glu-Gln-Ala-Pro-Glu-Asp-Gln-Gly-Pro-Gln-Arg-Glu-Pro-Tyr-Asn-Glu-Trp-Thr-Leu-Glu-
Leu-Leu-Glu-Glu-Leu-Lys-Ser-Glu-Ala-Val-Arg-His-Phe-Pro-Arg-Ile-Trp-Leu-His-Asn-Leu-Gly-
Gln-His-Ile-Tyr-Glu-Thr-Tyr-Gly-Asp-Thr-Trp-Ala-Gly-Val-Glu-Ala-Ile-Ile-Arg-Ile-Leu-Gln-Gln-
Leu-leu-Phe-Ile-His-Phe-Arg-Ile-Gly-Cys-Arg-His-Ser-Arg-Ile-Gly-Val-Thr-Arg-Gln-Arg-Arg-Ala-
Arg-Asn-Gly-Ala-
Ser-Arg-Ser-OHThe peptide was constructed on a TentaGel S-AC resin (0.20 mmol / gram) on an ABI 433. At the end of the synthesis, the FMOC protective group was split off, the resin was washed successively with dimethylformamide and methylene chloride and dried. The peptide was then cleaved from the resin in the manner described at the outset and then purified.
Molar mass: 11378 found 11381
H-Met-Glu-Gln-Ala-Pro-Glu-Asp-Gln-Gly-Pro-Gln-Arg-Gl-Pro-Tyr-Asn-Glu-Trp-Thr-Leu-Glu- Leu-Leu-Glu- Glu-Leu-Lys-Ser-Glu-Ala-Val-Arg-His-Phe-Pro-Arg-Ile-Trp-Leu-His-Asn-Leu-Gly- Gln-His-Ile-Tyr-Glu-Thr- Tyr-Gly-Asp-Thr-Trp-Ala-Gly-Val-Glu-Ala-Ile-Ile-Arg-Ile-Leu-Gln-Gln- Leu-leu-Phe-Ile-His-Phe-Arg-Ile- Gly-Cys-Arg-His-Ser-Arg-Ile-Gly-Val-Thr-Arg-Gln-Arg-Arg-Ala- Arg-Asn-Gly-Ala-
Ser-Arg-Ser-OH
Fig. 1 sVpr1-96 - Direkte Auftrennung im SDS-PAGE (A)
Immunpräzipitation vor SDS-PAGE (B), Fig. 1 sVpr1-96 - direct separation in SDS-PAGE (A)
Immunoprecipitation before SDS-PAGE (B),
Fig. 2 sVpr1-96 - Präparative Reinigung des Rohpeptids - HPLC-Chromatogramm, Figure 2 sVpr1-96 - Preparative purification of crude peptide -. HPLC chromatogram
Fig. 3 sVpr1-96 - Massenspektrum (% Int. und Molmasse), Fig. 3 sVpr1-96 - mass spectrum (% Int and molecular weight.)
Analog zu Beispielen 1 bis 3.
Molmasse: 5728 gef. 5728.8
H-Met-Glu-Gln-Ala-Pro-Glu-Asp-Gln-Gly-Pro-Gln-Arg-Glu-Pro-Tyr-Asn-Glu-Trp-Thr-Leu-Glu-
Leu-Leu-Glu-Glu-Leu-Lys-Ser-Glu-Ala-Val-Arg-His-Phe-Pro-Arg-Ile-Trp-Leu-His-Asn-Leu-Gly-
Gln-His-
Ile-Tyr-NH2 Analogous to Examples 1 to 3.
Molar mass: 5728 found 5728.8
H-Met-Glu-Gln-Ala-Pro-Glu-Asp-Gln-Gly-Pro-Gln-Arg-Gl-Pro-Tyr-Asn-Glu-Trp-Thr-Leu-Glu- Leu-Leu-Glu- Glu-Leu-Lys-Ser-Glu-Ala-Val-Arg-His-Phe-Pro-Arg-Ile-Trp-Leu-His-Asn-Leu-Gly- Gln-His-
Ile-Tyr-NH 2
Fig. 4 sVpr1-47 - Massenspektrum (% Int. und Molmasse) Fig. 4 sVpr1-47 - mass spectrum (% Int and molecular weight.)
Analog zu Beispielen 1 bis 3.
Glu-Thr-Tyr-Gly-Asp-Thr-Trp-Ala-Gly-Val-Glu Ala-Ile-Ile-Arg-Ile-Leu-Gln-Gln-Leu-leu-Phe-Ile-
His-Phe-Arg-Ile-Gly-Cys-Arg-His-Ser-Arg-Ile-Gly-Val-Thr-Arg-Gln-Arg-Arg-Ala-Arg-Asn-Gly-
Ala-Ser-Arg-Ser-OHAnalogous to Examples 1 to 3.
Glu-Thr-Tyr-Gly-Asp-Thr-Trp-Ala-Gly-Val-Glu Ala-Ile-Ile-Arg-Ile-Leu-Gln-Gln-Leu-leu-Phe-Ile-His-Phe-Arg -Ile-Gly-Cys-Arg-His-Ser-Arg-Ile-Gly-Val-Thr-Arg-Gln-Arg-Arg-Ala-Arg-Asn-Gly-Ala-Ser-Arg-Ser-OH
Analog zu Beispielen 1 bis 3.
H-Met-Glu-Gin-Ala-Pro-Glu-Asp-Gln-Gly-Pro-Gln-Argclu-Pro-Tyr-Asn-Glu-Trp-Thr-Leu-NH2 Analogous to Examples 1 to 3.
H-Met-Glu-Gin-Ala-Pro-Glu-Asp-Gln-Gly-Pro-Gln-Argclu-Pro-Tyr-Asn-Glu-Trp-Thr-Leu-NH 2
Fig. 5 sVpr1-20 - Massenspektrum (%Int. 10% = 111 mV [sum = 9505 mv] Profiles 1-85 Unsmoothed und Molmasse) Fig. 5 sVpr1-20 - mass spectrum (% Int. 10% = 111 mV [sum = 9505 mv] profiles 1-85 unsmoothed and molar mass)
Analog zu Beispielen 1 bis 3.
H-Met-Glu-Gln-Ala-Pro-Glu-Asp-Gln-Gly-Pro-Gln-Arg-Glu-Pro-Tyr-Asn-Glu-Trp-Thr-Leu-NH2 Analogous to Examples 1 to 3.
H-Met-Glu-Gln-Ala-Pro-Glu-Asp-Gln-Gly-Pro-Gln-Arg-Glu-Pro-Tyr-Asn-Glu-Trp-Thr-Leu-NH 2
Analog zu Beispielen 1 bis 3.
Wildtyp-Sequenz
H-Glu-Leu-Leu-Glu-Glu-Leu-Lys-Ser-Glu-Ala-Val-Arg-His-Phe-Asn-Arg-Ile-Trp-Leu-His-NH2 Analogous to Examples 1 to 3.
Wild type sequence
H-Glu-Leu-Leu-Glu-Glu-Leu-Lys-Ser-Glu-Ala-Val-Arg-His-Phe-Asn-Arg-Ile-Trp-Leu-His-NH 2
Fig. 6 sVpr21-40 - Massenspektrum (%Int. 10% = 335 mV [sum = 28541 mv] Profiles 1-85 Unsmoothed und Molmasse) Fig. 6 sVpr21-40 - mass spectrum (% Int. 10% = 335 mV [sum = 28541 mv] Profiles 1-85 Unsmoothed and Molar Mass)
Analog zu Beispielen 1 bis 3.
H-Glu-Leu-Leu-Glu-Glu-Leu-Lys-Ser-Glu-Ala-Val-Arg-His-Phe-Asn-Arg-Ile-Trp-Leu-His-NH2 Analogous to Examples 1 to 3.
H-Glu-Leu-Leu-Glu-Glu-Leu-Lys-Ser-Glu-Ala-Val-Arg-His-Phe-Asn-Arg-Ile-Trp-Leu-His-NH 2
Analog zu Beispielen 1 bis 3.
Gln-Arg-Glu-Pro-Tyr-Asn-Glu-Trp-Thr-Leu-Glu-Leu-Leu-Glu-Glu-Analogous to Examples 1 to 3.
Gln-Arg-Glu-Pro-Tyr-Asn-Glu-Trp-Thr-Leu-Glu-Leu-Leu-Glu-Glu-
Analog zu Beispielen 1 bis 3.
Asn-Leu-Gly-Gln-His-Ile-Tyr-Glu-Thr-Tyr-Gly-Asp-Thr-Trp-AlaAnalogous to Examples 1 to 3.
Asn-Leu-Gly-Gln-His-Ile-Tyr-Glu-Thr-Tyr-Gly-Asp-Thr-Trp-Ala
Analog zu Beispielen 1 bis 3.
Ile-Tyr-Glu-Thr-Tyr-Gly-Asp-Thr-Trp-Ala-Gly-Val-Glu-Ala-Ile-Analogous to Examples 1 to 3.
Ile-Tyr-Glu-Thr-Tyr-Gly-Asp-Thr-Trp-Ala-Gly-Val-Glu-Ala-Ile-
Analog zu Beispielen 1 bis 3.
Gly-Val-Glu-Ala-Ile-Ile-Arg-Ile-Leu-Gln-Gln-Leu-leu-Phe-IleAnalogous to Examples 1 to 3.
Gly-Val-Glu-Ala-Ile-Ile-Arg-Ile-Leu-Gln-Gln-Leu-leu-Phe-Ile
Analog zu Beispielen 1 bis 3.
Gln-Leu-Leu-Phe-Ile-His-Phe-Arg-Ile-Gly-Cys-Arg-His-Ser-ArgAnalogous to Examples 1 to 3.
Gln-Leu-Leu-Phe-Ile-His-Phe-Arg-Ile-Gly-Cys-Arg-His-Ser-Arg
Analog zu Beispielen 1 bis 3.
Cys-Arg-His-Ser-Arg-Ile-Gly-Val-Thr-Arg-Gln-Arg-Arg-Ala-Arg-Asn-Gly-Ala-Ser-Arg-Ser-OHAnalogous to Examples 1 to 3.
Cys-Arg-His-Ser-Arg-Ile-Gly-Val-Thr-Arg-Gln-Arg-Arg-Ala-Arg-Asn-Gly-Ala-Ser-Arg-Ser-OH
Adachi, A.; Gendelman, H.E.; König, S.; Folks, T.; Willey, R.L.; Rabson, A.; Martin, M.A.
(1986) Production of acquired immunodeflciency syndrome-associated retrovirus in human
and non-human cells transfected with an infectious molecular clone. J. Virol. 59: 284-291.
Arunagiri, C.; Macreadie, I; Hewish, D.; Azad, A. (1997) A C-terminal domain of HIV-1
accessory protein Vpr is involved in penetration, mitochondrial dysfunction and apoptosis of
human CD4+ lymphocytes. Apoptosis 2: 69-76.
Collman, J.W.; Balliet, J.W.; Greory, S.A.; Friedman, H.; Kolson, D.L; Nathanson, N.;
Srinivasan, A. (1992) An infectious molecular clone of an unusual macrophage-tropic and
highly cytopathic strain of human immunodeficiency virus type 1. J. Virol. 66 : 5717-5721.
Di Marzio, P.; Choe, S.; Ebright, M.; Knoblauch, R.; Landau, N.R. (1995) Mutational analysis
of cell cycle arrest, nuclear localization and virion packaging of human immunodeficiency virus
type 1 Vpr. J. Virol. 69: 7909-7916.
Kondo, E.; Göttlinger, H.G. (1996) A conserved LXXLF sequence is the major determinant in
p6gag required for the incoporation of human immunodeficiency virus type 1 Vpr. J. Virol.
70 : 159-164.
Kondo, E.; Mammano, F.; Cohen, E.A.; Göttlinger, H.G. (1995) The p6gag domain of human
immunodeficiency virus type 1 is sufficent for incorporation of Vpr into heterologous viral
particles. J. Virol.69: 2759-2764.
Lavallee, C.; Yao, X.J.; Ladha, A.; Göttlinger, H.G.; Haseltine, W.A.; Cohen, E.A. (1994)
Requirement of the Pr55gag precursor for incorporation of the Vpr product into human
immunodeficiency virus type 1 viral particles. J. Virol. 68: 1926-1934.
Levy, D.N.; Refaeli, Y.; Weiner, D.B. (1995) Extracellular Vpr protein increases cellular
permissiveness to human immunodeficiency virus type 1. Proc.Natl.Acad.Sci. USA 91:
10873-10877.
Lu, Y.-L.; Bennett, R.P.; Wills, J. W.; Gorelick, R.; Ratner, L. (1995) A leucine tripiet repeat
sequence (LXX)4 in p69a9 is important for Vpr incorporation into human immunodeficiency virus
type 1 particles. J. Virol. 69: 6873-6879.
Luo, Z.; Butcher, D.J.; Murali, R.; Srinivasan, A.; Huang, Z. (1998) Structural studies of
synthetic peptide fragments derived from the HIV-1 Vpr protein. Biochem. Biophys. Research
Communications 244: 732-736.
Macreadie, I.G.; Arunagiri, C.K.; Hewish, D.R.; White, J.F.; Azad, A A. (1996) Extracellular
addition of a domain of HIV-1 Vpr containing the amino acid sequence motif H(S/F)RIG
causes cell membrane permeabilization and death. Mol.Microbiol. 19: 1185-1192.
Adachi, A .; Gendelman, HE; King's.; Folks, T .; Willey, RL; Rabson, A .; Martin, MA (1986) Production of acquired immunodeficiency syndrome-associated retrovirus in human and non-human cells transfected with an infectious molecular clone. J. Virol. 59: 284-291.
Arunagiri, C .; Macreadie, I; Hewish, D .; Azad, A. (1997) A C-terminal domain of HIV-1 accessory protein Vpr is involved in penetration, mitochondrial dysfunction and apoptosis of human CD4 + lymphocytes. Apoptosis 2: 69-76.
Collman, JW; Balliet, JW; Greory, SA; Friedman, H .; Kolson, DL; Nathanson, N .; Srinivasan, A. (1992) An infectious molecular clone of an unusual macrophage-tropic and highly cytopathic strain of human immunodeficiency virus type 1. J. Virol. 66: 5717-5721.
Di Marzio, P .; Choe, S .; Ebright, M .; Knoblauch, R .; Landau, NR (1995) Mutational analysis of cell cycle arrest, nuclear localization and virion packaging of human immunodeficiency virus type 1 Vpr. J. Virol. 69: 7909-7916.
Kondo, E .; Göttlinger, HG (1996) A conserved LXXLF sequence is the major determinant in p6 gag required for the incoporation of human immunodeficiency virus type 1 Vpr. J. Virol. 70: 159-164.
Kondo, E .; Mammano, F .; Cohen, EA; Göttlinger, HG (1995) The p6 gag domain of human immunodeficiency virus type 1 is sufficent for incorporation of Vpr into heterologous viral particles. J. Virol. 69: 2759-2764.
Lavallee, C .; Yao, XJ; Ladha, A .; Göttlinger, HG; Haseltine, WA; Cohen, EA (1994) Requirement of the Pr55 gag precursor for incorporation of the Vpr product into human immunodeficiency virus type 1 viral particles. J. Virol. 68: 1926-1934.
Levy, DN; Refaeli, Y .; Weiner, DB (1995) Extracellular Vpr protein increases cellular permissiveness to human immunodeficiency virus type 1. Proc.Natl.Acad.Sci. USA 91: 10873-10877.
Lu, Y.-L .; Bennett, RP; Wills, JW; Gorelick, R .; Ratner, L. (1995) A leucine tripiet repeat sequence (LXX) 4 in p69a9 is important for Vpr incorporation into human immunodeficiency virus type 1 particles. J. Virol. 69: 6873-6879.
Luo, Z .; Butcher, DJ; Murali, R .; Srinivasan, A .; Huang, Z. (1998) Structural studies of synthetic peptide fragments derived from the HIV-1 Vpr protein. Biochem. Biophys. Research Communications 244: 732-736.
Macreadie, IG; Arunagiri, CK; Hewish, DR; White, JF; Azad, A A. (1996) Extracellular addition of a domain of HIV-1 Vpr containing the amino acid sequence motif H (S / F) RIG causes cell membrane permeabilization and death. Mol. Microbiol. 19: 1185-1192.
Macreadie, I.G.; Kirkpatrick, A.; Strike, P.M.; Azad, A.A. (1997) Cytocidal activities of HIV-1
Vpr and SACIP peptides bioassayed in yeast. Protein and Peptide Letters 4: 181-186.
Mahalingam, S.; Ayyavoo, V.; Patel, M.; Kieber-Emmons, T.; Weiner, D.B. (1997) Nuclear
import, virion incorporation, and cell cycle arrest/differentiation are mediated by distinct
functional domains of human immunodeficiency virus type 1 Vpr. J. Virol. 71: 6339-6347.
Mahalingam, S.; Collman, R.G.; Patel, M.; Monken, C.E.; Srinivasan, A. (1995a) Functional
analysis of HIV-1 Vpr: Identification of determinants essential for subcellular localization. Virol.
212: 331-339.
Mahalingam, S.; Khan, S.H.; Jabbar, M.A.; Monken, C.E.; Collman, R.G.; Srinivasan, A.
(1995b) Identification of residues in the N-terminal acidic domain of HIV-1 Vpr essential for
virion incorporation. Virol. 207 : 297-302.
Mahalingam, S.; Khan, S.H.; Murali, R.; Jabbar, M.A.; Monken, C.E.; Collman, R.G.;
Srinivasan, A. (1995c) Mutagenesis of the putative alpha-helical domain of the Vpr protein of
human immunodeficiency virus type 1: effect on stability and virion incorporation.
Proc.Natl.Acad.Sci. USA 92: 3794-3798.
Mahalingam, S.; Patel, M.; Collman, R.G.; Srinivasan, A. (1995d) The carboxy-terminal
domain is essential for stability and not for virion incorporation of HIV-1 Vpr into virus particles.
Virol. 214: 647-652.
Nie, Z.; Bergeron, D.; Subbramanian, R.A.; Yao, X.-J.; Checroune, F.; Rougeau, N.; Cohen,
E.A. (1998) The putative alpha helix 2 of human immunodeficiency virus type 1 Vpr contains a
determinanfi which is responsible for the nuclear translocalization of proviral DNA in growth
arrested cells. J. Virol. 73: 4104-4115.
Paxton, W.; Connor, R.I.; Landau, N.R. (1993) Incorporation of vpr into human
immunodeficiency virus type 1 virions: requirement for the p6 region of gag and mutational
analysis. J. Virol. 67: 7229-7237.
Piller, S.C.; Ewart, G.D.; Premkumar, A.; Cox, G.B.; Gage, P.W. (1996) Vpr protein of human
immunodeficiency virus type 1 forms cation-selective channels in planar lipid bilayers.
Proc.Natl.Acad.Sci. USA 93: 111-115.
Roques, B.P.; Morellet, N.; de Rocquigny, H.; Demene, H.; Schueler, W.; Jullian, N. (1997)
Structure, biological functions and inhibition of the HIV-1 proteins Vpr and NCp7. Biochimie
79: 673-680.
de Rocquigny, H.; Petitjean, P.; Tanchou, V.; Decimo, D.; Drouot, L.; Delaunay, T.; Darlix, J.-
L.; Roques, B.P. (1997) The zinc fingers of HIV nucleocapsid protein NCp7 direct interactions
with the viral regulatory protein Vpr. J. Biol. Chem. 272(49): 30753-30759.
Wang, B.; Ge, Y.C.; Palasanthiran, P.; Xiang, S.-H.; Ziegler, J.; Dwyer, D.E.; Randle, C.;
Dowton, D.; Cunningham, A.; Saksena, N.K. (1996) Gene defects clustered at the C
terminus of the vpr gene of HIV-1 in long-term nonprogressing mother and child pair: in vivo
evolution of vpr quasispecies in blood and plasma. Virol. 223: 224-232.
Wang, L.; Mukherjee, S.; Narayan, O; Zhao, L.-J. (1996) Characterization of a leucine-zipper
like domain in Vpr protein of human immunodeficiency virus type 1. Gene 178: 7-13.
Yao, S.; Azad, A.A.; Macreadie, I.G.; Norton, R.S. (1998) Helical structure of polypeptides
from the C-terminal halfof HIV-1 Vpr. Protein and Peptide Letters 5: 127-134.
Yao, X.-J.; Subbramanian, R.A.; Rougeau, N; Boisvert, F.; Bergeron, D.; Cohen, E.A. (1995)
Mutagenic analysis of human immunodeficiency virus type 1 Vpr: role of a predicted N-
terminal alpha-helical structure in Vpr nuclear localization and virion incorporation. J. Virol.
69: 7032-7044.
Zhao, L.J.; Mukherjee, S.; Narayan, O. (1994a) Biochemical mechanism of HIV-1 Vpr
function: specific interaction with a cellular protein. 1 Biol. Chem. 269: 15577-15582.
Zhao, L.J.; Wang, L.; Mukherjee, S.; Narayan, O. (1994b) Biochemical mechanism of HIV-1
Vpr function: oligomerization by the N-terminal domain. J. Biol. Chem. 269: 32131-32137.
Zhao, Y.; Cao, J.; O'Gorman, M. R.; Yu, M.; Yogev, R. (1996) Effect of human
immunodeficiency virus type 1 protein R (vpr) gene expression on basic cellular function of
fission yeast Schizosaccharomyces pombe. J. Virol. 70: 5821-5826.Macreadie, IG; Kirkpatrick, A .; Strike, PM; Azad, AA (1997) Cytocidal activities of HIV-1 Vpr and SACIP peptides bioassayed in yeast. Protein and Peptide Letters 4: 181-186.
Mahalingam, S .; Ayyavoo, V .; Patel, M .; Kieber-Emmons, T .; Weiner, DB (1997) Nuclear import, virion incorporation, and cell cycle arrest / differentiation are mediated by distinct functional domains of human immunodeficiency virus type 1 Vpr. J. Virol. 71: 6339-6347.
Mahalingam, S .; Collman, RG; Patel, M .; Monken, CE; Srinivasan, A. (1995a) Functional analysis of HIV-1 Vpr: Identification of determinants essential for subcellular localization. Virol. 212: 331-339.
Mahalingam, S .; Khan, SH; Jabbar, MA; Monken, CE; Collman, RG; Srinivasan, A. (1995b) Identification of residues in the N-terminal acidic domain of HIV-1 Vpr essential for virion incorporation. Virol. 207: 297-302.
Mahalingam, S .; Khan, SH; Murali, R .; Jabbar, MA; Monken, CE; Collman, RG; Srinivasan, A. (1995c) Mutagenesis of the putative alpha-helical domain of the Vpr protein of human immunodeficiency virus type 1: effect on stability and virion incorporation. Proc.Natl.Acad.Sci. USA 92: 3794-3798.
Mahalingam, S .; Patel, M .; Collman, RG; Srinivasan, A. (1995d) The carboxy-terminal domain is essential for stability and not for virion incorporation of HIV-1 Vpr into virus particles. Virol. 214: 647-652.
Never, Z .; Bergeron, D .; Subbramanian, RA; Yao, X.-J .; Checroune, F .; Rougeau, N .; Cohen, EA (1998) The putative alpha helix 2 of human immunodeficiency virus type 1 Vpr contains a determinanfi which is responsible for the nuclear translocalization of proviral DNA in growth arrested cells. J. Virol. 73: 4104-4115.
Paxton, W .; Connor, RI; Landau, NR (1993) Incorporation of vpr into human immunodeficiency virus type 1 virions: requirement for the p6 region of gag and mutational analysis. J. Virol. 67: 7229-7237.
Piller, SC; Ewart, GD; Premkumar, A .; Cox, GB; Gage, PW (1996) Vpr protein of human immunodeficiency virus type 1 forms cation-selective channels in planar lipid bilayers. Proc.Natl.Acad.Sci. USA 93: 111-115.
Roques, BP; Morellet, N .; de Rocquigny, H .; Demene, H .; Schueler, W .; Jullian, N. (1997) Structure, biological functions and inhibition of the HIV-1 proteins Vpr and NCp7. Biochimia 79: 673-680.
de Rocquigny, H .; Petitjean, P .; Tanchou, V .; Decimo, D .; Drouot, L .; Delaunay, T .; Darlix, J.-L .; Roques, BP (1997) The zinc fingers of HIV nucleocapsid protein NCp7 direct interactions with the viral regulatory protein Vpr. J. Biol. Chem. 272 (49): 30753-30759.
Wang, B .; Ge, YC; Palasanthiran, P .; Xiang, S.-H .; Ziegler, J .; Dwyer, DE; Randle, C .; Dowton, D .; Cunningham, A .; Saksena, NK (1996) Gene defects clustered at the C terminus of the vpr gene of HIV-1 in long-term nonprogressing mother and child pair: in vivo evolution of vpr quasispecies in blood and plasma. Virol. 223: 224-232.
Wang, L .; Mukherjee, S .; Narayan, O; Zhao, L.-J. (1996) Characterization of a leucine-zipper like domain in Vpr protein of human immunodeficiency virus type 1. Gene 178: 7-13.
Yao, S .; Azad, AA; Macreadie, IG; Norton, RS (1998) Helical structure of polypeptides from the C-terminal halfof HIV-1 Vpr. Protein and Peptide Letters 5: 127-134.
Yao, X.-J .; Subbramanian, RA; Rougeau, N; Boisvert, F .; Bergeron, D .; Cohen, EA (1995) Mutagenic analysis of human immunodeficiency virus type 1 Vpr: role of a predicted N-terminal alpha-helical structure in Vpr nuclear localization and virion incorporation. J. Virol. 69: 7032-7044.
Zhao, LJ; Mukherjee, S .; Narayan, O. (1994a) Biochemical mechanism of HIV-1 Vpr function: specific interaction with a cellular protein. 1 Biol. Chem. 269: 15577-15582.
Zhao, LJ; Wang, L .; Mukherjee, S .; Narayan, O. (1994b) Biochemical mechanism of HIV-1 Vpr function: oligomerization by the N-terminal domain. J. Biol. Chem. 269: 32131-32137.
Zhao, Y .; Cao, J .; O'Gorman, MR; Yu, M .; Yogev, R. (1996) Effect of human immunodeficiency virus type 1 protein R (vpr) gene expression on basic cellular function of fission yeast Schizosaccharomyces pombe. J. Virol. 70: 5821-5826.
Fig. 1 sVpr1-96 - Direkte Auftrennung im SDS-PAGE (A), Immunpräzipitation vor SDS-PAGE (B), Fig. 1 sVpr1-96 - Direct separation in SDS-PAGE (A), immunoprecipitation before SDS-PAGE (B),
Fig. 2 sVpr1-96 - Massenspektrum, Fig. 2 sVpr1-96 - mass spectrum
Fig. 3 sVpr1-96 - Chromatogramm, Fig. 3 sVpr1-96 - chromatogram
Fig. 4 sVpr1-47 - Massenspektrum, Fig. 4 sVpr1-47 - mass spectrum
Fig. 5 sVpr1-20 - Massenspektrum, Fig. 5 sVpr1-20 - mass spectrum
Fig. 6: sVpr21-40 - Massenspektrum. Fig. 6: sVpr21-40 - mass spectrum.
Claims (9)
- 1. 2.1. ein 96 Aminosäuren langes Vpr-Protein (sVpr1-96)
- 2. 2.2. ein 47 Aminosäuren langes N-terminales Peptid (sVpr1-47)
- 3. 2.3. ein 49 Aminosäuren langes C-terminales Peptid (sVpr48-96) sowie
- 4. 2.4. Fragmente dieser Peptide, zum Beispiel
- 1. 2.4.1. überlappende, etwa 15 Aminosäuren lange Peptide für die Epitop-Charakterisierung und isolelektrische Fokussierung
- 2. 2.4.2. etwa 20 Aminosäuren lange Peptide zur strukturellen und funktionellen Charakterisierung einzelner Domänen von Vpr, insbesondere
- 3. 2..2.1. die Peptide sVpr1-20 und
- 4. 2.4.2.2. sVpr21-40
- 1. 2.1. a 96 amino acid Vpr protein (sVpr1-96)
- 2. 2.2. a 47 amino acid long N-terminal peptide (sVpr1-47)
- 3. 2.3. a 49 amino acid long C-terminal peptide (sVpr48-96) as well
- 4. 2.4. Fragments of these peptides, for example
- 1. 2.4.1. overlapping, about 15 amino acid long peptides for epitope characterization and isolelectric focusing
- 2. 2.4.2. peptides about 20 amino acids long for the structural and functional characterization of individual domains of Vpr, in particular
- 3. 2..2.1. the peptides sVpr1-20 and
- 4. 2.4.2.2. sVpr21-40
- 1. 3.1. bei dem 96 Aminosäuren langen Vpr-Protein um
sVpr1-96
H-Met-Glu-Gln-Ala-Pro-Glu-Asp-Gln-Gly-Pro-Gln-Arg-Glu-Pro-Tyr-Asn-Glu-Trp-Thr-Leu- Glu-Leu-Leu-Glu-Glu-Leu-Lys-Ser-Glu-Ala-Val-Arg-His-Phe-Pro-Arg-Ile-Trp-Leu-His- Asn-Leu-Gly-Gln-His-Ile-Tyr-Glu-Thr-Tyr-Gly-Asp-Thr-Trp-Ala-Gly-Val-Glu-Ala-Ile lle-Arg-Ile-Leu-Gln-Gln-Leu-leu-Phe-Ile-His-Phe-Arg-Ile-Gly-Cys-Arg-His-Ser-Arg- Ile-Gly-Val-Thr-Arg-Gln-Arg-Arg-Ala-Arg-Asn-Gly-Ala-Ser-Arg-Ser-OH - 2. 3.2. bei dem 47 Aminosäuren langen N-terminalen Peptid um
sVpr1-47
H-Met-Glu-Gln-Ala-Pro-Glu-Asp-Gln-Gly-Pro-Gln-Arg-Glu-Pro-Tyr-Asn-Glu-Trp-Thr-Leu- Glu-Leu-Leu-Glu-Glu-Leu-Lys-Ser-Glu-Ala-Val-Arg-His-Phe-Pro-Arg-Ile-Trp-Leu-His- Asn-Leu-Gly-Gln-His-Ile-Tyr-NH2 - 3. 3.3. bei dem 49 Aminosäuren langen C-terminalen Peptid um
sVpr48-96
Glu-Thr-Tyr-Gly-Asp-Thr-Trp-Ala-Gly-Val-Glu-Ala-Ile-Ile-Arg-Ile-Leu-Gln-Gln-Leu leu-Phe-Ile-His-Phe-Arg-Ile-Gly-Cys-Arg-His-Ser-Arg-Ile-Gly-Val-Thr-Arg-Gln-Arg Arg-Ala-Arg-Asn-Gly-Ala-Ser-Arg-Ser-OH - 4. 3.4. bei den Fragmenten dieser Peptide um die etwa 15 Aminosäuren lange Peptide
- 1. 3.4.1. sVprl1-25
Gln-Arg-Glu-Pro-Tyr-Asn-Glu-Trp-Thr-Leu-Glu-Leu-Leu-Glu-Glu- - 2. 3.4.2. sVpr41-55
Asn-Leu-Gly-Gln-His-l le-Tyr-Glu-Thr-Tyr-Gly-Asp-Thr-Trp-Ala - 3. 3.4.3. sVpr46-60
Ile-Tyr-Glu-Thr-Tyr-Gly-Asp-Thr-Trp-Ala-Gly-Val-Glu-Ala-Ile- - 4. 3.4.4. sVpr56-70
Gly-Val-Glu-Ala-Ile-Ile-Arg-Ile-Leu-Gln-Gln-Leu-leu-Phe-Ile - 5. 3.4.5. sVpr66-80
Gln-Leu-leu-Phe-Ile-His-Phe-Arg-Ile-Gly-Cys-Arg-His-Ser-Arg - 6. 3.4.6. sVpr76-96
Cys-Arg-His-Ser-Arg-Ile-Gly-Val-Thr-Arg-Gln-Arg-Arg-Ala-Arg-Asn-Gly-Ala-Ser-Arg-Ser-OH
- 1. 3.4.1. sVprl1-25
- 5. 3.5. bei den etwa 20 Aminosäuren langen Peptiden um
- 1. 3.5.1. die Peptide sVpr1-20 als
sVpr1-20(Asn5,10,14)
H-Met-Glu-Gln-Ala-Asn-Glu-Asp-Gln-Gly-Asn-Gln-Arg-Glu-Asn-Tyr-Asn-Glu-Trp-Thr-Leu-NH2 und - 2. 3.5.2. sVpr21-40 als
sVpr 21-40(Asn35)
H-Glu-Leu-Leu-Glu-Glu-Leu-Lys-Ser-Glu-Ala-Val-Arg-His-Phe-Asn-Arg-Ile-Trp-Leu-His-NH2
- 1. 3.5.1. die Peptide sVpr1-20 als
- 1. 3.1. around the 96 amino acid Vpr protein
sVpr1-96
H-Met-Glu-Gln-Ala-Pro-Glu-Asp-Gln-Gly-Pro-Gln-Arg-Gl-Pro-Tyr-Asn-Glu-Trp-Thr-Leu- Glu-Leu-Leu-Glu- Glu-Leu-Lys-Ser-Glu-Ala-Val-Arg-His-Phe-Pro-Arg-Ile-Trp-Leu-His- Asn-Leu-Gly-Gln-His-Ile-Tyr-Glu-Thr- Tyr-Gly-Asp-Thr-Trp-Ala-Gly-Val-Glu-Ala-Ile lle-Arg-Ile-Leu-Gln-Gln-Leu-leu-Phe-Ile-His-Phe-Arg-Ile-Gly -Cys-Arg-His-Ser-Arg-Ile-Gly-Val-Thr-Arg-Gln-Arg-Arg-Ala-Arg-Asn-Gly-Ala-Ser-Arg-Ser-OH - 2. 3.2. around the 47 amino acid long N-terminal peptide
sVpr1-47
H-Met-Glu-Gln-Ala-Pro-Glu-Asp-Gln-Gly-Pro-Gln-Arg-Gl-Pro-Tyr-Asn-Glu-Trp-Thr-Leu- Glu-Leu-Leu-Glu- Glu-Leu-Lys-Ser-Glu-Ala-Val-Arg-His-Phe-Pro-Arg-Ile-Trp-Leu-His-Asn-Leu-Gly-Gln-His-Ile-Tyr-NH 2 - 3. 3.3. around the 49 amino acid long C-terminal peptide
sVpr48-96
Glu-Thr-Tyr-Gly-Asp-Thr-Trp-Ala-Gly-Val-Glu-Ala-Ile-Ile-Arg-Ile-Leu-Gln-Gln-Leu leu-Phe-Ile-His-Phe-Arg -Ile-Gly-Cys-Arg-His-Ser-Arg-Ile-Gly-Val-Thr-Arg-Gln-Arg Arg-Ala-Arg-Asn-Gly-Ala-Ser-Arg-Ser-OH - 4. 3.4. in the fragments of these peptides around the 15 amino acid peptides
- 1. 3.4.1. sVprl1-25
Gln-Arg-Glu-Pro-Tyr-Asn-Glu-Trp-Thr-Leu-Glu-Leu-Leu-Glu-Glu- - 2. 3.4.2. sVpr41-55
Asn-Leu-Gly-Gln-His-l le-Tyr-Glu-Thr-Tyr-Gly-Asp-Thr-Trp-Ala - 3. 3.4.3. sVpr46-60
Ile-Tyr-Glu-Thr-Tyr-Gly-Asp-Thr-Trp-Ala-Gly-Val-Glu-Ala-Ile- - 4. 3.4.4. sVpr56-70
Gly-Val-Glu-Ala-Ile-Ile-Arg-Ile-Leu-Gln-Gln-Leu-leu-Phe-Ile - 5. 3.4.5. sVpr66-80
Gln-Leu-leu-Phe-Ile-His-Phe-Arg-Ile-Gly-Cys-Arg-His-Ser-Arg - 6. 3.4.6. sVpr76-96
Cys-Arg-His-Ser-Arg-Ile-Gly-Val-Thr-Arg-Gln-Arg-Arg-Ala-Arg-Asn-Gly-Ala-Ser-Arg-Ser-OH
- 1. 3.4.1. sVprl1-25
- 5.3.5. with the approximately 20 amino acid long peptides
- 1.3.5.1. the peptides sVpr1-20 as
sVpr1-20 (Asn 5,10,14 )
H-Met-Glu-Gln-Ala-Asn-Glu-Asp-Gln-Gly-Asn-Gln-Arg-Glu-Asn-Tyr-Asn-Glu-Trp-Thr-Leu-NH 2 and - 2. 3.5.2. sVpr21-40 as sVpr 21-40 (Asn35)
H-Glu-Leu-Leu-Glu-Glu-Leu-Lys-Ser-Glu-Ala-Val-Arg-His-Phe-Asn-Arg-Ile-Trp-Leu-His-NH 2
- 1.3.5.1. the peptides sVpr1-20 as
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE19908752A DE19908752A1 (en) | 1999-02-19 | 1999-02-19 | New synthetic peptides from the Vpr protein of human immune deficiency virus, useful e.g. for therapy and diagnosis, have good solubility in water |
PCT/DE2000/000525 WO2000049038A2 (en) | 1999-02-19 | 2000-02-19 | Synthetic peptide of regulatory virus protein r (vpr) of human immunodeficiency virus type 1 (hiv-1) and the utilization thereof |
JP2000599775A JP2002540768A (en) | 1999-02-19 | 2000-02-19 | Synthetic peptide of human immunodeficiency virus type 1 (HIV-1) viral regulatory protein R (Vpr) and its application |
EP00918674A EP1155035A2 (en) | 1999-02-19 | 2000-02-19 | Synthetic peptide of regulatory virus protein r (vpr) of human immunodeficiency virus type 1 (hiv-1) and the utilization thereof |
US09/913,927 US6984486B1 (en) | 1999-02-19 | 2000-02-19 | Synthetic peptide of regulatory virus protein R (VPR) of human immunodeficiency virus type 1 (HIV-1) and the utilization thereof |
CA002356390A CA2356390A1 (en) | 1999-02-19 | 2001-08-17 | Synthetic peptide of regulatory virus protein r (vpr) of human immunodeficiency virus type 1 (hiv-1) and the utilization thereof |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE19908752A DE19908752A1 (en) | 1999-02-19 | 1999-02-19 | New synthetic peptides from the Vpr protein of human immune deficiency virus, useful e.g. for therapy and diagnosis, have good solubility in water |
CA002356390A CA2356390A1 (en) | 1999-02-19 | 2001-08-17 | Synthetic peptide of regulatory virus protein r (vpr) of human immunodeficiency virus type 1 (hiv-1) and the utilization thereof |
Publications (1)
Publication Number | Publication Date |
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DE19908752A1 true DE19908752A1 (en) | 2000-08-31 |
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Family Applications (1)
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DE19908752A Withdrawn DE19908752A1 (en) | 1999-02-19 | 1999-02-19 | New synthetic peptides from the Vpr protein of human immune deficiency virus, useful e.g. for therapy and diagnosis, have good solubility in water |
Country Status (1)
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DE (1) | DE19908752A1 (en) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995026361A1 (en) * | 1994-03-25 | 1995-10-05 | Biomolecular Research Institute Ltd. | Vpr AND Vpx PROTEINS OF HIV |
WO1996007741A1 (en) * | 1994-09-07 | 1996-03-14 | Universite De Montreal | Protein targeting into hiv virions based on hiv-1 vpr fusion molecules |
-
1999
- 1999-02-19 DE DE19908752A patent/DE19908752A1/en not_active Withdrawn
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1995026361A1 (en) * | 1994-03-25 | 1995-10-05 | Biomolecular Research Institute Ltd. | Vpr AND Vpx PROTEINS OF HIV |
WO1996007741A1 (en) * | 1994-09-07 | 1996-03-14 | Universite De Montreal | Protein targeting into hiv virions based on hiv-1 vpr fusion molecules |
Non-Patent Citations (1)
Title |
---|
J. Acquired Immune Defic.Syndr. 1994, 7 (7), S. 635-40 * |
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