Die posttranslationale Modifikation durch kleine Ubiquitin-ähnliche Modifikatoren (SUMO) wird mit einer wachsenden Zahl regulatorischer Funktionen in verschiedenen zellulären Prozessen in Verbindung gebracht. Die biochemische Untersuchung der zugrunde liegenden molekularen Mechanismen hinkt jedoch hinterher, da es schwierig ist, ausreichende Mengen rekombinanter SUMOylierter Proteine herzustellen. Hier stellen wir zwei neu entwickelte Zweikomponenten-Vektorsysteme für die Expression und Reinigung von SUMO-modifizierten Zielproteinen in Escherichia coli vor . Ein System besteht aus einem Vektor für die SUMO-Konjugation, der humane SUMO-aktivierende (SAE1/SAE2) und konjugierende (Ubc9) Enzyme zusammen mit His 6 exprimiert-getaggte SUMO1, 2 oder 3, die mit häufig verwendeten Expressionskonstrukten für jedes interessierende Gen kombiniert werden können.
Um die SUMOylierung von Zielen zu erleichtern, die normalerweise eine SUMO-E3-Ligase für eine effiziente Modifikation benötigen, wird ein zweites System entwickelt, um das Zielprotein als Fusion mit dem humanen SUMO-konjugierenden Enzym Ubc9 zu exprimieren und so das Fehlen einer potenziellen SUMO-Ligase zu kompensieren. Wir demonstrieren die Leistungsfähigkeit dieser Systeme durch SUMOylierung von zwei DNA-Reparaturproteinen, der Thymin-DNA-Glykosylase (TDG) und XRCC1, und beschreiben Reinigungsschemata für SUMOylierte Proteine in nativer und aktiver Form. Diese SUMO-Toolbox erleichtert die Produktion und Reinigung rekombinanter SUMO-modifizierter Zielproteine für die Funktions- und Strukturanalyse „in der Zelle“ und „im Extrakt“.
Einführun
Die posttranslationale Modifikation durch Ubiquitin-ähnliche Polypeptide, sogenannte UBLs, betrifft eine Vielzahl von Proteinen und reguliert dadurch eine Vielzahl zellulärer Prozesse . Die SUMO-Peptide (kleine Ubiquitin-ähnliche Modifikatoren) stellen eine prominente Unterfamilie der UBLs dar und existieren in Säugerzellen in vier verschiedenen Isoformen (SUMO1, SUMO2, SUMO3 und SUMO4), die jeweils von einem anderen Gen kodiert werden. Obwohl sich diese SUMOs bis zu einem gewissen Grad in ihren Aminosäuresequenzen unterscheiden – SUMO2 und SUMO3 haben eine Sequenzidentität von 97 % miteinander und etwa 50 % mit SUMO1 – zeigen sie alle eine hohe 3D-Strukturähnlichkeit. SUMOs werden, wie alle UBLs, durch eine Sequenz von enzymatischen Reaktionen, die denen der Ubiquitin-Konjugation ähneln, an ihre Zielproteine gebunden , wobei ein heterodimeres aktivierendes Enzym E1 (SAE1/SAE2), ein einzelnes konjugierendes Enzym E2 (Ubc9 ) und in einigen Fällen eine E3-Proteinligase ( 1A ).
SUMO selbst wird zunächst als Vorläuferpeptid synthetisiert, das dann durch eine SUMO-spezifische Isopeptidase (Sentrin-spezifische Proteasen; SenPs) getrimmt wird, um ein internes Glycin-Glycin (GG)-Motiv am C-Terminus freizulegen. Die Carboxylgruppe dieses reifen SUMO-Peptids wird dann ATP-abhängig über einen Thioester an einen Cysteinrest in SAE2 gebunden. Anschließend wird das aktivierte SUMO auf einen Cysteinrest des SUMO-konjugierenden Enzyms Ubc9übertragen . Ubc9 kann Substratproteine direkt erkennen und die Bildung einer Peptidbindung katalysieren, die die C-terminale Carboxylgruppe von SUMO und eine ε-Aminogruppe eines Ziel-Lysins innerhalb des SUMOylierungs-Konsensusmotivs ΨKxE (Ψ, hydrophober Rest; x, irgendein Rest) des Substratproteins.
Although research into protein SUMOylation has identified a large number of targets over recent years, there is limited insight into the functional consequences of the modification.
Investigations into the immediate biochemical and structural impact of SUMO modification have been a challenge due to difficulties in producing SUMO-modified proteins in sufficient amounts and homogeneity. Enrichment of native endogenous SUMOylated proteins by cell fractionation is generally limited by the low abundance of such proteins and the action of efficient SUMO proteases . In vitro modification of enriched target proteins with recombinant SUMOylation enzymes is a more promising approach, typically yielding mixtures of modified and unmodified target proteins contaminated with the SUMOylation enzymes, hence requiring subsequent purification steps. Also, co-expression of SUMO targets with SUMO1 and SUMO-activating and -conjugating enzymes of different origin (human, mouse, Xenopus laevis) in E.coli was shown to produce SUMO-modified protein , but in our experience, the yields of specifically modified proteins were often poor, impeding efficient purification and subsequent biochemical analysis
To streamline the production of homogeneously SUMOylated recombinant proteins for biochemical and structural studies, we set out to establish an optimized and versatile SUMOylation system, coupling efficient SUMO-conjugation with affinity purification of modified target proteins. We designed and experimentally validated two alternative two-component vector systems for simultaneous expression of mature SUMO1, 2 or 3 polypeptides, SUMO-E1, SUMO-E2 and a target protein of interest in E.coli.
In contrast to previous approaches, we used SUMOylation enzymes of human origin only, physically separated the SAE1 and SAE2 E1 subunits and added a protease-cleavable His6-tag to the SUMOs to facilitate purification of modified protein. To overcome a possible rate limitation by the absence of an appropriate SUMO-E3 ligase in the E.coli system, one system is designed to express the target protein as fusion to the SUMO-conjugating enzyme Ubc9, a strategy that was successfully applied for SUMOylation of ectopically expressed p53 and STAT1 in HEK293, HeLa, COS-7 and CHO cells . We evaluated these newly developed vector systems with the DNA base excision repair (BER) enzymes thymine DNA glycosylase (TDG) and XRCC1.
Recombinant Human CTRB1 Protein, Untagged, E.coli-50mg |
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QP10565-50mg | EnQuireBio | 50mg | 563 EUR |
Recombinant E.coli LACTB Protein, Untagged, E.coli-50mg |
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QP12522-50mg | EnQuireBio | 50mg | 3655 EUR |
Recombinant other Urease Protein, Untagged, E.coli-50mg |
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QP10907-50mg | EnQuireBio | 50mg | 1043 EUR |
Recombinant other Atosiban Protein, Untagged, E.coli-50mg |
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QP11102-50mg | EnQuireBio | 50mg | 218 EUR |
Recombinant other Exenatide Protein, Untagged, E.coli-50mg |
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QP11816-50mg | EnQuireBio | 50mg | 2358 EUR |
Recombinant other Terlipressin Protein, Untagged, E.coli-50mg |
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QP13723-50mg | EnQuireBio | 50mg | 363 EUR |
Recombinant Bovine HTF Protein, Untagged, E.coli-50mg |
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QP12351-50mg | EnQuireBio | 50mg | 155 EUR |
Recombinant Human Trypsin-2 Protein, Untagged, E.coli-50mg |
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QP10899-50mg | EnQuireBio | 50mg | 563 EUR |
Recombinant Rat Carboxypeptidase B Protein, Untagged, E.coli-50mg |
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QP10544-50mg | EnQuireBio | 50mg | 517 EUR |
Recombinant Bovine ATF Protein, Untagged, -50mg |
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QP11095-50mg | EnQuireBio | 50mg | 155 EUR |
Recombinant Yeast IDH1 Protein, Untagged, Yeast-50mg |
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QP10670-50mg | EnQuireBio | 50mg | 1043 EUR |
Recombinant Multi Species ACTH Protein, Untagged, -50mg |
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QP10950-50mg | EnQuireBio | 50mg | 553 EUR |
Recombinant other HSA Recombinant Protein, Untagged, Rice-50mg |
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QP12317-50mg | EnQuireBio | 50mg | 363 EUR |
Recombinant Human Collagen-III Protein, Untagged, Pichia Pastoris-50mg |
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QP11479-50mg | EnQuireBio | 50mg | 926 EUR |
Recombinant Porcine Trypsin Protein, Untagged, Pichia Pastoris-50mg |
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QP10897-50mg | EnQuireBio | 50mg | 962 EUR |
Recombinant other HSA Protein, Untagged, Pichia Pastoris-50mg |
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QP12314-50mg | EnQuireBio | 50mg | 201 EUR |
Recombinant other HSA Lipid free Protein, Untagged, Rice-50mg |
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QP12316-50mg | EnQuireBio | 50mg | 363 EUR |
Recombinant Human CTRB1 Protein, Untagged, E.coli-10mg |
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QP10565-10mg | EnQuireBio | 10mg | 201 EUR |
Recombinant Human CTRB1 Protein, Untagged, E.coli-1mg |
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QP10565-1mg | EnQuireBio | 1mg | 155 EUR |
Mitomycin C, 50mg |
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R5007-50mg | ACTGene | 1427 EUR | |
Human Chymotrypsinogen B1 (CTRB1) ELISA Kit |
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DLR-CTRB1-Hu-48T | DL Develop | 48T | 517 EUR |
Human Chymotrypsinogen B1 (CTRB1) ELISA Kit |
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DLR-CTRB1-Hu-96T | DL Develop | 96T | 673 EUR |
Human Chymotrypsinogen B1 (CTRB1) ELISA Kit |
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RD-CTRB1-Hu-48Tests | Reddot Biotech | 48 Tests | 521 EUR |
Human Chymotrypsinogen B1 (CTRB1) ELISA Kit |
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RD-CTRB1-Hu-96Tests | Reddot Biotech | 96 Tests | 723 EUR |
Human Chymotrypsinogen B1 (CTRB1) ELISA Kit |
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RDR-CTRB1-Hu-48Tests | Reddot Biotech | 48 Tests | 544 EUR |
Human Chymotrypsinogen B1 (CTRB1) ELISA Kit |
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RDR-CTRB1-Hu-96Tests | Reddot Biotech | 96 Tests | 756 EUR |
CTRB1 Recombinant Protein (Human) |
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RP008329 | ABM | 100 ug | Ask for price |
Recombinant E.coli G6PD Protein, Untagged, E.coli-10ug |
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QP10622-10ug | EnQuireBio | 10ug | 155 EUR |
Recombinant E.coli G6PD Protein, Untagged, E.coli-1mg |
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QP10622-1mg | EnQuireBio | 1mg | 1859 EUR |
Recombinant E.coli G6PD Protein, Untagged, E.coli-50ug |
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QP10622-50ug | EnQuireBio | 50ug | 201 EUR |
Recombinant E.coli SlyD Protein, Untagged, E.coli-1mg |
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QP10865-1mg | EnQuireBio | 1mg | 2757 EUR |
Recombinant E.coli SlyD Protein, Untagged, E.coli-20ug |
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QP10865-20ug | EnQuireBio | 20ug | 201 EUR |
Recombinant E.coli SlyD Protein, Untagged, E.coli-5ug |
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QP10865-5ug | EnQuireBio | 5ug | 155 EUR |
Recombinant E.coli TRXR Protein, Untagged, E.coli-1mg |
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QP10894-1mg | EnQuireBio | 1mg | 2312 EUR |
Recombinant E.coli TRXR Protein, Untagged, E.coli-25ug |
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QP10894-25ug | EnQuireBio | 25ug | 201 EUR |
Recombinant E.coli TRXR Protein, Untagged, E.coli-5ug |
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QP10894-5ug | EnQuireBio | 5ug | 155 EUR |
Recombinant E.coli LACTB Protein, Untagged, E.coli-10mg |
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QP12522-10mg | EnQuireBio | 10mg | 962 EUR |
Recombinant E.coli LACTB Protein, Untagged, E.coli-1mg |
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QP12522-1mg | EnQuireBio | 1mg | 201 EUR |
CTRB1 Recombinant Protein (Mouse) |
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RP126617 | ABM | 100 ug | Ask for price |
CTRB1 Recombinant Protein (Rat) |
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RP196709 | ABM | 100 ug | Ask for price |
DiA: (50mg) |
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60013 | Biotium | 50MG | 210 EUR |
Recombinant Human IHH Protein, Untagged, E.coli-100ug |
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QP10186-ec-100ug | EnQuireBio | 100ug | 952 EUR |
Recombinant Human IHH Protein, Untagged, E.coli-250ug |
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QP10186-ec-250ug | EnQuireBio | 250ug | 1623 EUR |
Recombinant Human IHH Protein, Untagged, E.coli-25ug |
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QP10186-ec-25ug | EnQuireBio | 25ug | 290 EUR |
Recombinant Human IHH Protein, Untagged, E.coli-5ug |
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QP10186-ec-5ug | EnQuireBio | 5ug | 154 EUR |
Recombinant Human XCL1 Protein, Untagged, E.coli-100ug |
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QP10221-ec-100ug | EnQuireBio | 100ug | 988 EUR |
Recombinant Human XCL1 Protein, Untagged, E.coli-20ug |
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QP10221-ec-20ug | EnQuireBio | 20ug | 290 EUR |
Recombinant Human XCL1 Protein, Untagged, E.coli-250ug |
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QP10221-ec-250ug | EnQuireBio | 250ug | 1731 EUR |
Recombinant Human XCL1 Protein, Untagged, E.coli-5ug |
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QP10221-ec-5ug | EnQuireBio | 5ug | 154 EUR |
Recombinant Human CCL28 Protein, Untagged, E.coli-100ug |
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QP10243-ec-100ug | EnQuireBio | 100ug | 988 EUR |
Recombinant Human CCL28 Protein, Untagged, E.coli-20ug |
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QP10243-ec-20ug | EnQuireBio | 20ug | 290 EUR |
Recombinant Human CCL28 Protein, Untagged, E.coli-250ug |
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QP10243-ec-250ug | EnQuireBio | 250ug | 1731 EUR |
Recombinant Human CCL28 Protein, Untagged, E.coli-5ug |
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QP10243-ec-5ug | EnQuireBio | 5ug | 154 EUR |
Recombinant Human FAM19A2 Protein, Untagged, E.coli-20ug |
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QP10244-ec-20ug | EnQuireBio | 20ug | 290 EUR |
Recombinant Human FAM19A2 Protein, Untagged, E.coli-5ug |
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QP10244-ec-5ug | EnQuireBio | 5ug | 154 EUR |
Recombinant Human IL5 Protein, Untagged, E.coli-100ug |
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QP10312-ec-100ug | EnQuireBio | 100ug | 1396 EUR |
Recombinant Human IL5 Protein, Untagged, E.coli-10ug |
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QP10312-ec-10ug | EnQuireBio | 10ug | 290 EUR |
Recombinant Human IL5 Protein, Untagged, E.coli-250ug |
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QP10312-ec-250ug | EnQuireBio | 250ug | 2457 EUR |
Recombinant Human IL5 Protein, Untagged, E.coli-2ug |
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QP10312-ec-2ug | EnQuireBio | 2ug | 154 EUR |
Recombinant Human TSLP Protein, Untagged, E.coli-100ug |
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QP10350-ec-100ug | EnQuireBio | 100ug | 1678 EUR |
Recombinant Human TSLP Protein, Untagged, E.coli-10ug |
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QP10350-ec-10ug | EnQuireBio | 10ug | 290 EUR |
Recombinant Human TSLP Protein, Untagged, E.coli-250ug |
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QP10350-ec-250ug | EnQuireBio | 250ug | 2956 EUR |
Recombinant Human TSLP Protein, Untagged, E.coli-2ug |
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QP10350-ec-2ug | EnQuireBio | 2ug | 154 EUR |
Recombinant Human LIF Protein, Untagged, E.coli-100ug |
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QP10360-ec-100ug | EnQuireBio | 100ug | 934 EUR |
Recombinant Human LIF Protein, Untagged, E.coli-250ug |
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QP10360-ec-250ug | EnQuireBio | 250ug | 1623 EUR |
Recombinant Human LIF Protein, Untagged, E.coli-25ug |
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QP10360-ec-25ug | EnQuireBio | 25ug | 290 EUR |
Recombinant Human LIF Protein, Untagged, E.coli-5ug |
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QP10360-ec-5ug | EnQuireBio | 5ug | 154 EUR |
Recombinant Human Plasminogen Protein, Untagged, E.coli-100ug |
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QP10364-ec-100ug | EnQuireBio | 100ug | 362 EUR |
Recombinant Human Plasminogen Protein, Untagged, E.coli-10ug |
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QP10364-ec-10ug | EnQuireBio | 10ug | 154 EUR |
Recombinant Human Plasminogen Protein, Untagged, E.coli-250ug |
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QP10364-ec-250ug | EnQuireBio | 250ug | 590 EUR |
Recombinant Human Plasminogen Protein, Untagged, E.coli-50ug |
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QP10364-ec-50ug | EnQuireBio | 50ug | 290 EUR |
Recombinant Human TNFRSF11A Protein, Untagged, E.coli-100ug |
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QP10370-ec-100ug | EnQuireBio | 100ug | 290 EUR |
Recombinant Human TNFRSF11A Protein, Untagged, E.coli-20ug |
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QP10370-ec-20ug | EnQuireBio | 20ug | 154 EUR |
Recombinant Human TNFRSF11A Protein, Untagged, E.coli-250ug |
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QP10370-ec-250ug | EnQuireBio | 250ug | 544 EUR |
Recombinant Human TNFSF18 Protein, Untagged, E.coli-100ug |
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QP10373-ec-100ug | EnQuireBio | 100ug | 1115 EUR |
Recombinant Human TNFSF18 Protein, Untagged, E.coli-20ug |
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QP10373-ec-20ug | EnQuireBio | 20ug | 290 EUR |
Recombinant Human TNFSF18 Protein, Untagged, E.coli-250ug |
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QP10373-ec-250ug | EnQuireBio | 250ug | 2312 EUR |
Recombinant Human TNFSF18 Protein, Untagged, E.coli-5ug |
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QP10373-ec-5ug | EnQuireBio | 5ug | 154 EUR |
Recombinant Human FGF4 Protein, Untagged, E.coli-25ug |
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QP10378-ec-25ug | EnQuireBio | 25ug | 290 EUR |
Recombinant Human FGF4 Protein, Untagged, E.coli-5ug |
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QP10378-ec-5ug | EnQuireBio | 5ug | 154 EUR |
Recombinant Human FGF9 Protein, Untagged, E.coli-100ug |
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QP10380-ec-100ug | EnQuireBio | 100ug | 1024 EUR |
Recombinant Human FGF9 Protein, Untagged, E.coli-20ug |
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QP10380-ec-20ug | EnQuireBio | 20ug | 290 EUR |
Recombinant Human FGF9 Protein, Untagged, E.coli-250ug |
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QP10380-ec-250ug | EnQuireBio | 250ug | 1750 EUR |
Recombinant Human FGF9 Protein, Untagged, E.coli-5ug |
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QP10380-ec-5ug | EnQuireBio | 5ug | 154 EUR |
Recombinant Human IGFBP3 Protein, Untagged, E.coli-100ug |
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QP10386-ec-100ug | EnQuireBio | 100ug | 988 EUR |
Recombinant Human IGFBP3 Protein, Untagged, E.coli-250ug |
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QP10386-ec-250ug | EnQuireBio | 250ug | 1731 EUR |
Recombinant Human IGFBP3 Protein, Untagged, E.coli-25ug |
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QP10386-ec-25ug | EnQuireBio | 25ug | 290 EUR |
Recombinant Human IGFBP3 Protein, Untagged, E.coli-5ug |
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QP10386-ec-5ug | EnQuireBio | 5ug | 154 EUR |
Recombinant Human Epigen Protein, Untagged, E.coli-100ug |
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QP10396-ec-100ug | EnQuireBio | 100ug | 988 EUR |
Recombinant Human Epigen Protein, Untagged, E.coli-250ug |
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QP10396-ec-250ug | EnQuireBio | 250ug | 1731 EUR |
Recombinant Human Epigen Protein, Untagged, E.coli-25ug |
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QP10396-ec-25ug | EnQuireBio | 25ug | 290 EUR |
Recombinant Human Epigen Protein, Untagged, E.coli-5ug |
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QP10396-ec-5ug | EnQuireBio | 5ug | 154 EUR |
Recombinant Human Amphiregulin Protein, Untagged, E.coli-100ug |
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QP10399-ec-100ug | EnQuireBio | 100ug | 525 EUR |
Recombinant Human Amphiregulin Protein, Untagged, E.coli-10ug |
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QP10399-ec-10ug | EnQuireBio | 10ug | 154 EUR |
Recombinant Human Amphiregulin Protein, Untagged, E.coli-250ug |
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QP10399-ec-250ug | EnQuireBio | 250ug | 898 EUR |
Recombinant Human Amphiregulin Protein, Untagged, E.coli-50ug |
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QP10399-ec-50ug | EnQuireBio | 50ug | 290 EUR |
Recombinant Human NTF4 Protein, Untagged, E.coli-100ug |
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QP10412-ec-100ug | EnQuireBio | 100ug | 1678 EUR |
Recombinant Human NTF4 Protein, Untagged, E.coli-10ug |
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QP10412-ec-10ug | EnQuireBio | 10ug | 290 EUR |
Recombinant Human NTF4 Protein, Untagged, E.coli-250ug |
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QP10412-ec-250ug | EnQuireBio | 250ug | 2956 EUR |
Recombinant Human NTF4 Protein, Untagged, E.coli-2ug |
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QP10412-ec-2ug | EnQuireBio | 2ug | 154 EUR |
TDG is a well-studied SUMO target; SUMO modification of TDG was shown to effect conformational changes that promote enzymatic turnover and may also regulate the subcellular localization. XRCC1 acts as central scaffold factor in BER and was identified as a putative SUMO target in an in vitro screening approach. It was found SUMOylated in HeLa cells following heat shock treatment, but the function of this modification remains to be elucidated.
We assessed the efficiency and specificity of SUMO modification of these proteins in our recombinant systems as well as the proficiency of the purification procedure to generate biologically active SUMO proteins. Finally, we provide guidance to optimize the experimental setup and conditions for the SUMOylation of any target protein in bacteria, discussing the use of the different SUMOylation vectors and expression strategies for “in-cell” or “in-extract” SUMOylation.