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

QP10565-1mg EnQuireBio 1mg 186 EUR

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

QP10565-10mg EnQuireBio 10mg 241.2 EUR

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

QP12522-50mg EnQuireBio 50mg 4386 EUR

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

QP10899-50mg EnQuireBio 50mg 675.6 EUR

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

QP12351-50mg EnQuireBio 50mg 186 EUR

Recombinant other Urease Protein, Untagged, E.coli-50mg

QP10907-50mg EnQuireBio 50mg 1251.6 EUR

Recombinant other Atosiban Protein, Untagged, E.coli-50mg

QP11102-50mg EnQuireBio 50mg 261.6 EUR

Recombinant other Exenatide Protein, Untagged, E.coli-50mg

QP11816-50mg EnQuireBio 50mg 2829.6 EUR

Recombinant other Terlipressin Protein, Untagged, E.coli-50mg

QP13723-50mg EnQuireBio 50mg 435.6 EUR

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

QP10544-50mg EnQuireBio 50mg 620.4 EUR

Recombinant Bovine ATF Protein, Untagged, -50mg

QP11095-50mg EnQuireBio 50mg 186 EUR

Recombinant Human OT Protein, Untagged, E.coli-5mg

QP12937-5mg EnQuireBio 5mg 186 EUR

Recombinant Human OT Protein, Untagged, E.coli-25mg

QP12937-25mg EnQuireBio 25mg 241.2 EUR

Recombinant Human OT Protein, Untagged, E.coli-100mg

QP12937-100mg EnQuireBio 100mg 544.8 EUR

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

QP13766-1mg EnQuireBio 1mg 1154.4 EUR

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

QP13828-1mg EnQuireBio 1mg 4386 EUR

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

QP2835-1mg EnQuireBio 1mg 3189.6 EUR

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

QP5308-1mg EnQuireBio 1mg 3189.6 EUR

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

QP12228-1mg EnQuireBio 1mg 3308.4 EUR

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

QP10855-1mg EnQuireBio 1mg 1034.4 EUR

Recombinant Human BD5 Protein, Untagged, E.coli-1mg

QP11149-1mg EnQuireBio 1mg 3308.4 EUR

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

QP11198-1mg EnQuireBio 1mg 6301.2 EUR

Recombinant Human CA3 Protein, Untagged, E.coli-1mg

QP11248-1mg EnQuireBio 1mg 2774.4 EUR

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

QP11435-1mg EnQuireBio 1mg 6301.2 EUR

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

QP12775-1mg EnQuireBio 1mg 1154.4 EUR

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

QP12840-1mg EnQuireBio 1mg 6301.2 EUR

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

QP12920-1mg EnQuireBio 1mg 186 EUR

Recombinant Human OCT Protein, Untagged, E.coli-5mg

QP12920-5mg EnQuireBio 5mg 337.2 EUR

Recombinant Human IL5 Protein, Untagged, E.coli-1mg

QP1177-1mg EnQuireBio 1mg 4983.6 EUR

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

QP11784-10ug EnQuireBio 10ug 663.6 EUR

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

QP11513-1mg EnQuireBio 1mg 469.2 EUR

Recombinant Human CRP Protein, Untagged, E.coli-5mg

QP11513-5mg EnQuireBio 5mg 1698 EUR

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

QP10792-1mg EnQuireBio 1mg 4963.2 EUR

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

QP10811-1mg EnQuireBio 1mg 4963.2 EUR

Recombinant Human OCT Protein, Untagged, E.coli-25mg

QP12920-25mg EnQuireBio 25mg 1154.4 EUR

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

QP11784-2ug EnQuireBio 2ug 294 EUR

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

QP11784-5ug EnQuireBio 5ug 403.2 EUR

Recombinant Human TRH Protein, Untagged, E.coli-100mg

QP13814-100mg EnQuireBio 100mg 381.6 EUR

Recombinant Human TRH Protein, Untagged, E.coli-250mg

QP13814-250mg EnQuireBio 250mg 675.6 EUR

Recombinant Human TRH Protein, Untagged, E.coli-500mg

QP13814-500mg EnQuireBio 500mg 980.4 EUR

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

QP10186-ec-25ug EnQuireBio 25ug 348 EUR

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

QP10312-ec-10ug EnQuireBio 10ug 348 EUR

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

QP13695-1mg EnQuireBio 1mg 3308.4 EUR

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

QP13766-20ug EnQuireBio 20ug 186 EUR

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

QP13825-1mg EnQuireBio 1mg 3308.4 EUR

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

QP13828-20ug EnQuireBio 20ug 241.2 EUR

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

QP1387-ec-10ug EnQuireBio 10ug 184.8 EUR

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

QP1387-ec-50ug EnQuireBio 50ug 348 EUR

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

QP5220-1mg EnQuireBio 1mg 5942.4 EUR

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

QP5221-1mg EnQuireBio 1mg 5942.4 EUR

Recombinant Human IL25 Protein, Untagged, E.coli-1mg

QP5273-1mg EnQuireBio 1mg 3427.2 EUR

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

QP5289-1mg EnQuireBio 1mg 5942.4 EUR

Recombinant Human FGF8 Protein, Untagged, E.coli-1mg

QP5301-1mg EnQuireBio 1mg 3427.2 EUR

Recombinant Human FGF4 Protein, Untagged, E.coli-1mg

QP5343-1mg EnQuireBio 1mg 3427.2 EUR

Recombinant Human NTF4 Protein, Untagged, E.coli-1mg

QP5345-1mg EnQuireBio 1mg 5942.4 EUR

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

QP5371-1mg EnQuireBio 1mg 3427.2 EUR

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

QP5373-1mg EnQuireBio 1mg 4027.2 EUR

Recombinant Human GDF5 Protein, Untagged, E.coli-1mg

QP5375-1mg EnQuireBio 1mg 1633.2 EUR

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

QP5382-1mg EnQuireBio 1mg 3427.2 EUR

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

QP12205-1mg EnQuireBio 1mg 4386 EUR

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

QP12228-20ug EnQuireBio 20ug 241.2 EUR

Recombinant Human KLF4 Protein, Untagged, E.coli-1mg

QP12498-1mg EnQuireBio 1mg 6301.2 EUR

Recombinant Human RLN3 Protein, Untagged, E.coli-1mg

QP10859-1mg EnQuireBio 1mg 3308.4 EUR

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

QP10873-1mg EnQuireBio 1mg 5680.8 EUR

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

QP10875-1mg EnQuireBio 1mg 1263.6 EUR

Recombinant Human ATF3 Protein, Untagged, E.coli-1mg

QP11098-1mg EnQuireBio 1mg 3308.4 EUR

Recombinant Human ATF4 Protein, Untagged, E.coli-1mg

QP11099-1mg EnQuireBio 1mg 6301.2 EUR

Recombinant Human BAG1 Protein, Untagged, E.coli-1mg

QP11124-1mg EnQuireBio 1mg 2774.4 EUR

Recombinant Human BCL2 Protein, Untagged, E.coli-1mg

QP11142-1mg EnQuireBio 1mg 3308.4 EUR

Recombinant Human BD5 Protein, Untagged, E.coli-20ug

QP11149-20ug EnQuireBio 20ug 241.2 EUR

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

QP11198-10ug EnQuireBio 10ug 241.2 EUR

Recombinant Human CA3 Protein, Untagged, E.coli-20ug

QP11248-20ug EnQuireBio 20ug 241.2 EUR

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

QP11271-1mg EnQuireBio 1mg 2230.8 EUR

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

QP11435-10ug EnQuireBio 10ug 241.2 EUR

Recombinant Human CKS2 Protein, Untagged, E.coli-1mg

QP11439-1mg EnQuireBio 1mg 3308.4 EUR

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

QP11451-1mg EnQuireBio 1mg 6301.2 EUR

Recombinant Human MIA2 Protein, Untagged, E.coli-1mg

QP12694-1mg EnQuireBio 1mg 3308.4 EUR

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

QP12775-20ug EnQuireBio 20ug 186 EUR

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

QP12840-10ug EnQuireBio 10ug 241.2 EUR

Recombinant Human NME2 Protein, Untagged, E.coli-1mg

QP12855-1mg EnQuireBio 1mg 2774.4 EUR

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

QP12877-1mg EnQuireBio 1mg 6301.2 EUR

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

QP12938-1mg EnQuireBio 1mg 4287.6 EUR

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

QP13083-1mg EnQuireBio 1mg 2774.4 EUR

Recombinant Human RAC1 Protein, Untagged, E.coli-1mg

QP13249-1mg EnQuireBio 1mg 2230.8 EUR

Recombinant Human RAC2 Protein, Untagged, E.coli-1mg

QP13250-1mg EnQuireBio 1mg 4386 EUR

Recombinant Human RBP4 Protein, Untagged, E.coli-1mg

QP13283-1mg EnQuireBio 1mg 4386 EUR

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

QP13313-1mg EnQuireBio 1mg 2230.8 EUR

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

QP13378-1mg EnQuireBio 1mg 2230.8 EUR

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

QP13536-1mg EnQuireBio 1mg 2350.8 EUR

Recombinant Human ST13 Protein, Untagged, E.coli-1mg

QP13601-1mg EnQuireBio 1mg 2230.8 EUR

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

QP11787-1mg EnQuireBio 1mg 6301.2 EUR

Recombinant Human FDX1 Protein, Untagged, E.coli-1mg

QP11864-1mg EnQuireBio 1mg 3667.2 EUR

Recombinant Human FEN1 Protein, Untagged, E.coli-1mg

QP11866-1mg EnQuireBio 1mg 4386 EUR

Recombinant Human FGF6 Protein, Untagged, E.coli-1mg

QP11872-1mg EnQuireBio 1mg 3308.4 EUR

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

QP11946-50ug EnQuireBio 50ug 1393.2 EUR

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

QP12004-1mg EnQuireBio 1mg 5680.8 EUR

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

QP12006-1mg EnQuireBio 1mg 3308.4 EUR

Recombinant Human GYG1 Protein, Untagged, E.coli-1mg

QP12070-1mg EnQuireBio 1mg 3667.2 EUR

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

QP11514-20ug EnQuireBio 20ug 241.2 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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