Rekombinantes menschliches CTRB1-Protein, ohne Tag, E. coli-50 mg

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

QP10565-50mg EnQuireBio 50mg 675.6 EUR

Recombinant E.coli LACTB Protein, Untagged, E.coli-50mg

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Recombinant Bovine HTF Protein, Untagged, E.coli-50mg

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Recombinant Human Trypsin-2 Protein, Untagged, E.coli-50mg

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Recombinant Rat Carboxypeptidase B Protein, Untagged, E.coli-50mg

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Recombinant Bovine ATF Protein, Untagged, -50mg

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Recombinant other HSA Recombinant Protein, Untagged, Rice-50mg

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Recombinant Human Collagen-III Protein, Untagged, Pichia Pastoris-50mg

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Recombinant Porcine Trypsin Protein, Untagged, Pichia Pastoris-50mg

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Recombinant other HSA Protein, Untagged, Pichia Pastoris-50mg

QP12314-50mg EnQuireBio 50mg 241.2 EUR

Recombinant other HSA Lipid free Protein, Untagged, Rice-50mg

QP12316-50mg EnQuireBio 50mg 435.6 EUR

Recombinant Human CTRB1 Protein, Untagged, E.coli-10mg

QP10565-10mg EnQuireBio 10mg 241.2 EUR

Recombinant Human CTRB1 Protein, Untagged, E.coli-1mg

QP10565-1mg EnQuireBio 1mg 186 EUR

Mitomycin C, 50mg

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Human Chymotrypsinogen B1 (CTRB1) ELISA Kit

DLR-CTRB1-Hu-48T DL Develop 48T 620.4 EUR

Human Chymotrypsinogen B1 (CTRB1) ELISA Kit

DLR-CTRB1-Hu-96T DL Develop 96T 807.6 EUR

Human Chymotrypsinogen B1 (CTRB1) ELISA Kit

RD-CTRB1-Hu-48Tests Reddot Biotech 48 Tests 625.2 EUR

Human Chymotrypsinogen B1 (CTRB1) ELISA Kit

RD-CTRB1-Hu-96Tests Reddot Biotech 96 Tests 867.6 EUR

Human Chymotrypsinogen B1 (CTRB1) ELISA Kit

RDR-CTRB1-Hu-48Tests Reddot Biotech 48 Tests 652.8 EUR

Human Chymotrypsinogen B1 (CTRB1) ELISA Kit

RDR-CTRB1-Hu-96Tests Reddot Biotech 96 Tests 907.2 EUR

Goat Anti-Mouse IgG Fc, 50MG

A008-50MG Arbor Assays 50MG 938 EUR

Goat Anti-Rabbit IgG Fc, 50MG

A009-50MG Arbor Assays 50MG 938 EUR

Donkey Anti-Sheep IgG (H&L), 50MG

A010-50MG Arbor Assays 50MG 938 EUR

CTRB1 Recombinant Protein (Human)

RP008329 ABM 100 ug Ask for price

CTRB1 Recombinant Protein (Rat)

RP196709 ABM 100 ug Ask for price

CTRB1 Recombinant Protein (Mouse)

RP126617 ABM 100 ug Ask for price

CTRB1 Chymotrypsinogen-B1, Human Recombinant Protein

PROTP17538 BosterBio Regular: 10mg 380.4 EUR

Recombinant E.coli LACTB Protein, Untagged, E.coli-10mg

QP12522-10mg EnQuireBio 10mg 1154.4 EUR

Recombinant E.coli LACTB Protein, Untagged, E.coli-1mg

QP12522-1mg EnQuireBio 1mg 241.2 EUR

Recombinant E.coli G6PD Protein, Untagged, E.coli-10ug

QP10622-10ug EnQuireBio 10ug 186 EUR

Recombinant E.coli G6PD Protein, Untagged, E.coli-1mg

QP10622-1mg EnQuireBio 1mg 2230.8 EUR

Recombinant E.coli G6PD Protein, Untagged, E.coli-50ug

QP10622-50ug EnQuireBio 50ug 241.2 EUR

Recombinant E.coli SlyD Protein, Untagged, E.coli-1mg

QP10865-1mg EnQuireBio 1mg 3308.4 EUR

Recombinant E.coli SlyD Protein, Untagged, E.coli-20ug

QP10865-20ug EnQuireBio 20ug 241.2 EUR

Recombinant E.coli SlyD Protein, Untagged, E.coli-5ug

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Recombinant E.coli TRXR Protein, Untagged, E.coli-1mg

QP10894-1mg EnQuireBio 1mg 2774.4 EUR

Recombinant E.coli TRXR Protein, Untagged, E.coli-25ug

QP10894-25ug EnQuireBio 25ug 241.2 EUR

Recombinant E.coli TRXR Protein, Untagged, E.coli-5ug

QP10894-5ug EnQuireBio 5ug 186 EUR

Recombinant Human MICA Protein, Untagged, E.coli-10ug

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Recombinant Human MICA Protein, Untagged, E.coli-50ug

QP10172-ec-50ug EnQuireBio 50ug 348 EUR

Recombinant Human MICB Protein, Untagged, E.coli-100ug

QP10173-ec-100ug EnQuireBio 100ug 1413.6 EUR

Recombinant Human MICB Protein, Untagged, E.coli-10ug

QP10173-ec-10ug EnQuireBio 10ug 184.8 EUR

Recombinant Human MICB Protein, Untagged, E.coli-250ug

QP10173-ec-250ug EnQuireBio 250ug 2480.4 EUR

Recombinant Human MICB Protein, Untagged, E.coli-50ug

QP10173-ec-50ug EnQuireBio 50ug 348 EUR

Recombinant Human IHH Protein, Untagged, E.coli-100ug

QP10186-ec-100ug EnQuireBio 100ug 1142.4 EUR

Recombinant Human IHH Protein, Untagged, E.coli-250ug

QP10186-ec-250ug EnQuireBio 250ug 1947.6 EUR

Recombinant Human IHH Protein, Untagged, E.coli-25ug

QP10186-ec-25ug EnQuireBio 25ug 348 EUR

Recombinant Human IHH Protein, Untagged, E.coli-5ug

QP10186-ec-5ug EnQuireBio 5ug 184.8 EUR

Recombinant Human XCL1 Protein, Untagged, E.coli-100ug

QP10221-ec-100ug EnQuireBio 100ug 1185.6 EUR

Recombinant Human XCL1 Protein, Untagged, E.coli-20ug

QP10221-ec-20ug EnQuireBio 20ug 348 EUR

Recombinant Human XCL1 Protein, Untagged, E.coli-250ug

QP10221-ec-250ug EnQuireBio 250ug 2077.2 EUR

Recombinant Human XCL1 Protein, Untagged, E.coli-5ug

QP10221-ec-5ug EnQuireBio 5ug 184.8 EUR

Recombinant Human CCL28 Protein, Untagged, E.coli-100ug

QP10243-ec-100ug EnQuireBio 100ug 1185.6 EUR

Recombinant Human CCL28 Protein, Untagged, E.coli-20ug

QP10243-ec-20ug EnQuireBio 20ug 348 EUR

Recombinant Human CCL28 Protein, Untagged, E.coli-250ug

QP10243-ec-250ug EnQuireBio 250ug 2077.2 EUR

Recombinant Human CCL28 Protein, Untagged, E.coli-5ug

QP10243-ec-5ug EnQuireBio 5ug 184.8 EUR

Recombinant Human FAM19A2 Protein, Untagged, E.coli-20ug

QP10244-ec-20ug EnQuireBio 20ug 348 EUR

Recombinant Human FAM19A2 Protein, Untagged, E.coli-5ug

QP10244-ec-5ug EnQuireBio 5ug 184.8 EUR

Recombinant Human IL5 Protein, Untagged, E.coli-100ug

QP10312-ec-100ug EnQuireBio 100ug 1675.2 EUR

Recombinant Human IL5 Protein, Untagged, E.coli-10ug

QP10312-ec-10ug EnQuireBio 10ug 348 EUR

Recombinant Human IL5 Protein, Untagged, E.coli-250ug

QP10312-ec-250ug EnQuireBio 250ug 2948.4 EUR

Recombinant Human IL5 Protein, Untagged, E.coli-2ug

QP10312-ec-2ug EnQuireBio 2ug 184.8 EUR

Recombinant Human TSLP Protein, Untagged, E.coli-100ug

QP10350-ec-100ug EnQuireBio 100ug 2013.6 EUR

Recombinant Human TSLP Protein, Untagged, E.coli-10ug

QP10350-ec-10ug EnQuireBio 10ug 348 EUR

Recombinant Human TSLP Protein, Untagged, E.coli-250ug

QP10350-ec-250ug EnQuireBio 250ug 3547.2 EUR

Recombinant Human TSLP Protein, Untagged, E.coli-2ug

QP10350-ec-2ug EnQuireBio 2ug 184.8 EUR

Recombinant Human LIF Protein, Untagged, E.coli-100ug

QP10360-ec-100ug EnQuireBio 100ug 1120.8 EUR

Recombinant Human LIF Protein, Untagged, E.coli-250ug

QP10360-ec-250ug EnQuireBio 250ug 1947.6 EUR

Recombinant Human LIF Protein, Untagged, E.coli-25ug

QP10360-ec-25ug EnQuireBio 25ug 348 EUR

Recombinant Human LIF Protein, Untagged, E.coli-5ug

QP10360-ec-5ug EnQuireBio 5ug 184.8 EUR

Recombinant Human Plasminogen Protein, Untagged, E.coli-100ug

QP10364-ec-100ug EnQuireBio 100ug 434.4 EUR

Recombinant Human Plasminogen Protein, Untagged, E.coli-10ug

QP10364-ec-10ug EnQuireBio 10ug 184.8 EUR

Recombinant Human Plasminogen Protein, Untagged, E.coli-250ug

QP10364-ec-250ug EnQuireBio 250ug 708 EUR

Recombinant Human Plasminogen Protein, Untagged, E.coli-50ug

QP10364-ec-50ug EnQuireBio 50ug 348 EUR

Recombinant Human TNFRSF11A Protein, Untagged, E.coli-100ug

QP10370-ec-100ug EnQuireBio 100ug 348 EUR

Recombinant Human TNFRSF11A Protein, Untagged, E.coli-20ug

QP10370-ec-20ug EnQuireBio 20ug 184.8 EUR

Recombinant Human TNFRSF11A Protein, Untagged, E.coli-250ug

QP10370-ec-250ug EnQuireBio 250ug 652.8 EUR

Recombinant Human TNFSF18 Protein, Untagged, E.coli-100ug

QP10373-ec-100ug EnQuireBio 100ug 1338 EUR

Recombinant Human TNFSF18 Protein, Untagged, E.coli-20ug

QP10373-ec-20ug EnQuireBio 20ug 348 EUR

Recombinant Human TNFSF18 Protein, Untagged, E.coli-250ug

QP10373-ec-250ug EnQuireBio 250ug 2774.4 EUR

Recombinant Human TNFSF18 Protein, Untagged, E.coli-5ug

QP10373-ec-5ug EnQuireBio 5ug 184.8 EUR

Recombinant Human FGF4 Protein, Untagged, E.coli-25ug

QP10378-ec-25ug EnQuireBio 25ug 348 EUR

Recombinant Human FGF4 Protein, Untagged, E.coli-5ug

QP10378-ec-5ug EnQuireBio 5ug 184.8 EUR

Recombinant Human FGF9 Protein, Untagged, E.coli-100ug

QP10380-ec-100ug EnQuireBio 100ug 1228.8 EUR

Recombinant Human FGF9 Protein, Untagged, E.coli-20ug

QP10380-ec-20ug EnQuireBio 20ug 348 EUR

Recombinant Human FGF9 Protein, Untagged, E.coli-250ug

QP10380-ec-250ug EnQuireBio 250ug 2100 EUR

Recombinant Human FGF9 Protein, Untagged, E.coli-5ug

QP10380-ec-5ug EnQuireBio 5ug 184.8 EUR

Recombinant Human IGFBP3 Protein, Untagged, E.coli-100ug

QP10386-ec-100ug EnQuireBio 100ug 1185.6 EUR

Recombinant Human IGFBP3 Protein, Untagged, E.coli-250ug

QP10386-ec-250ug EnQuireBio 250ug 2077.2 EUR

Recombinant Human IGFBP3 Protein, Untagged, E.coli-25ug

QP10386-ec-25ug EnQuireBio 25ug 348 EUR

Recombinant Human IGFBP3 Protein, Untagged, E.coli-5ug

QP10386-ec-5ug EnQuireBio 5ug 184.8 EUR

Recombinant Human Epigen Protein, Untagged, E.coli-100ug

QP10396-ec-100ug EnQuireBio 100ug 1185.6 EUR

Recombinant Human Epigen Protein, Untagged, E.coli-250ug

QP10396-ec-250ug EnQuireBio 250ug 2077.2 EUR

Recombinant Human Epigen Protein, Untagged, E.coli-25ug

QP10396-ec-25ug EnQuireBio 25ug 348 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.

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