Pgex 6p 1

Manufactured by Merck Group

Sourced in Germany

The PGEX-6P-1 is a plasmid vector used for the expression of recombinant proteins in Escherichia coli. It contains a GST (Glutathione S-Transferase) tag, which facilitates the purification of the expressed protein using affinity chromatography. The vector also includes a tac promoter for inducible expression and a multiple cloning site for the insertion of the target gene.

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Lab products found in correlation

Pgex 6p 1, by GE Healthcare (1 mentions) Yeast extract, by BD (1 mentions) Kanamycin, by Merck Group (1 mentions) Bacto peptone, by BD (1 mentions) Spectinomycin, by Duchefa Biochemie (1 mentions) Ampicillin, by Duchefa Biochemie (1 mentions) Bamhi, by Merck Group (1 mentions) Ab236632, by Abcam (1 mentions) Ab111947, by Abcam (1 mentions) Phusion polymerase, by New England Biolabs (1 mentions) Pgex 6p, by Merck Group (1 mentions)

Spelling variants of this product

PGEX-6p-1 or pET-28a vector (2 citations) PGEX-6P (1 citations)

6 protocols using pgex 6p 1

Purification of Recombinant G Protein Subunits

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Gβ₁γ₁ was purified from bovine
retina as described previously.^{60 (link)} Recombinant
Gβ₁γ₂ and Gβ₅γ₂ were expressed
in Sf9 cells and purified via a His₆-tagged Gγ₂ using nickel-nitrilotriacetic acid affinity chromatography
(Sigma-Aldrich, St. Louis, MO). Human Gγ subunit cDNAs for Gγ₄, Gγ₅, Gγ₇, Gγ₁₁, and Gγ₁₃ were subcloned by polymerase
chain reaction from pcDNA3.1+ clones (Missouri S&T cDNA Resource
Center) into pGEX-6p-1 (GE Healthcare). The sequences of the resultant
vectors, hGgamma(_x).pGEX-6p-1, were verified,
and GST fusion proteins were expressed in Rosetta Competent Cells
(EMD Millipore). After induction with 1 mM IPTG followed by a 4 h
incubation at 37 °C, the resultant Gγ proteins were batch
purified using Glutathione Sepahrose 4 Fast Flow (GE Healthcare) and
eluted with 10 mM reduced free acid glutathione (Calbiochem).

Betke K.M., Rose K.L., Friedman D.B., Baucum AJ I.I., Hyde K., Schey K.L, & Hamm H.E. (2014). Differential Localization of G Protein βγ Subunits. Biochemistry, 53(14), 2329-2343.

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Bacterial Strains and Plasmids for Genetic Engineering

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All the bacterial strains and plasmids used in this study are listed in Table 1. The A. tumefaciens EHA105 strain was grown in YEP (10 g yeast extract (BD, Sparks, MD, USA), 10 g Bacto peptone (BD, Sparks, MD, USA), and 5 g NaCl/L, pH 7.0 with spectinomycin 100 µg/mL (Duchefa Biochemie, Shanghai, China)) at 28 °C in a shaker incubator (180 rpm). DH5α, BW25113, and BL21(DE3) of E. coli strains were used for cloning, cell toxicity, and protein expression assays. pBAD24, pET28a (BBI Life Sciences, Novagen, Darmstadt, Germany), and pGEX 6p-1 (Merck, St. Louis, MO, USA) plasmids were used in this study. The antibiotics for E. coli ampicillin (Duchefa Biochemie, Haarlem, Netherlands) and kanamycin (Merck, St. Louis, MO, USA) were used at concentrations of 100 µg/mL and 50 µg/mL, respectively.

Choi W., Yamaguchi Y., Park J.Y., Park S.H., Lee H.W., Lim B.K., Otto M., Inouye M., Yoon M.H, & Park J.H. (2021). Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens. Microorganisms, 9(5), 1107.

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Structural Characterization of SARS-CoV-2 Mpro

Cited 2 times

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The M^pro coding sequence was codon-optimized for expression in Escherichia coli and synthesized by Integrated DNA Technologies. The M^pro expression construct used for crystallization comprises an N-terminal glutathione S-transferase region, an M^pro autocleavage site, the M^pro coding sequence, a hybrid cleavage site recognizable by 3C Human Rhinovirus (HRV) protease, and a C-terminal 6-histidine tag (72 (link)). The overall construct was flanked by In-Fusion compatible ends for insertion into Bam HI–Xho I cleaved pGEX-6P-1 (Sigma). Protein expression, purification, and crystallization were carried out in similar conditions to those previously described by Douangamath et al. (73 (link)). Specifically, crystals were obtained from 0.1 M MES (pH 6.5), 15 PEG4K, and 5% dimethyl sulfoxide (DMSO) using drop ratios of 0.15 μl of protein, 0.3 μl of reservoir solution, and 0.05 μl of seed stock.

Chenthamarakshan V., Hoffman S.C., Owen C.D., Lukacik P., Strain-Damerell C., Fearon D., Malla T.R., Tumber A., Schofield C.J., Duyvesteyn H.M., Dejnirattisai W., Carrique L., Walter T.S., Screaton G.R., Matviiuk T., Mojsilovic A., Crain J., Walsh M.A., Stuart D.I, & Das P. (2023). Accelerating drug target inhibitor discovery with a deep generative foundation model. Science Advances, 9(25), eadg7865.

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Direct Binding of CENPN and CREB

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This assay was used to detect whether CENPN could directly bind to the target protein CREB. GST-CENPN and HA-CREB genes were first cloned and synthesized and then inserted into pGEX-6P-1 (Sigma, GE28-9546-48). After insert ligation, transformation into recipient cells and clonal expression, the protein samples containing 500 μg GST (control group) or GST-CENPN (experimental group) were added into glutathione-agarose resin (Thermo Scientific, 16100) and mixed for 3 h, respectively. Five hundred micrograms of HA-CREB protein were added to the control and experimental groups and then mixed overnight. The two groups of samples were centrifuged, and an appropriate amount of protein loading buffer was added and then incubated at 100°C for 5 min. Finally, WB experiments were performed with anti-GST (abcam, ab111947) and anti-HA (abcam, ab236632) antibodies for GST: GST-CENPN and HA-CREB, respectively.

Wang B.R., Han J.B., Jiang Y., Xu S., Yang R., Kong Y.G., Tao Z.Z., Hua Q.Q., Zou Y, & Chen S.M. (2024). CENPN suppresses autophagy and increases paclitaxel resistance in nasopharyngeal carcinoma cells by inhibiting the CREB-VAMP8 signaling axis. Autophagy, 20(2), 329-348.

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Expression and Purification of SARS-CoV-2 Proteases

Cited 2 times

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The M^pro coding sequence was codon optimised for expression in E. coli and synthesised by Integrated DNA technologies (IDT). The M^pro expression construct used for crystallization comprises an N-terminal GST region, an M^pro autocleavage site, the M^pro coding sequence, a hybrid cleavage site recognizable by 3C HRV protease and a C-terminal 6-Histidine tag^{54 (link)}. The overall construct was flanked by In-Fusion compatible ends for insertion into BamHI-XhoI cleaved pGEX-6P-1 (Sigma). An additional M^pro construct was generated with an extended 10-Histidine tag, for enhanced binding to the sensor surface in SPR assays. This construct was amplified by PCR from the above version, with the C-terminal primer incorporating a further 4-Histidines. The resulting amplicon was then inserted into BamHI-XhoI cleaved pGEX-6P-1 by In-Fusion cloning.
The PL^pro expression construct was similarly optimised and synthesised and comprised an N-terminal 10 Histidine tag followed by the PL^pro sequence (Nsp3 region E746-K1060). This was then directly inserted into NcoI-HindIII digested pOPINF via In-Fusion compatible ends. pOPINF was a gift from Ray Owens (University of Oxford)^{55 (link)} (Addgene plasmid # 26042 ; http://n2t.net/addgene:26042 ; RRID:Addgene_26042).

Redhead M.A., Owen C.D., Brewitz L., Collette A.H., Lukacik P., Strain-Damerell C., Robinson S.W., Collins P.M., Schäfer P., Swindells M., Radoux C.J., Hopkins I.N., Fearon D., Douangamath A., von Delft F., Malla T.R., Vangeel L., Vercruysse T., Thibaut J., Leyssen P., Nguyen T.T., Hull M., Tumber A., Hallett D.J., Schofield C.J., Stuart D.I., Hopkins A.L, & Walsh M.A. (2021). Bispecific repurposed medicines targeting the viral and immunological arms of COVID-19. Scientific Reports, 11, 13208.

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Cloning and Mutagenesis of BRD4 Bromodomains

Cited 1 time

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Human BRD4-BD1 (aa 44–168) and BRD4-BD2 (aa 349–461) coding sequences were amplified by PCR from pF:hBRD4(1–722)-11d DNA template^{78 (link)} using Phusion polymerase (NEB, M0530L) and primer pairs 5′-CGCGGA-TCCAACCCCCCGCCCCCAGAGACCTCCAAC-3′ and 5′-CCGCTCGAGTCATTCTTCTGTGGGTAGCTCATTTATTTT-3′ for BD1 and 5′-CGCGGATCCAAGGTCTCGGAGCAGCTCA-3′ and 5′-TTCCCGGGTCTAGATCAAGGCTCGTCCGGC-ATCTT-3′ for BD2. The PCR fragments were then digested with BamHI and XhoI sites (for BD1) or BamHI and SmaI sites (for BD2) and inserted into a linearized pGEX-6P-1 (Sigma-Aldrich GE28–9546-48) with the corresponding enzymes to generate pGEX-6P-1-hBRD4-BD1 and pGEX-6P-1-hBRD4-BD2, respectively. Generation of BD1 E151A mutation was done by site-directed mutagenesis using primer pair 5′-GTCTTAATGGCAGCAGCTCTGGAAAAG-3′ and 5′-CTTTTCCAGAGCTGCTGCCATTAAGAC and pGEX-6P-1-hBRD4-BD1 template for PCR amplification at 98°C for 1 min, followed by 25 cycles of 98°C for 45 s, 55°C for 45 s, and 68°C for 12 min. The PCR products were then digested with DpnI at 37°C for 2 h to degrade the template plasmid with the mutated construct named pGEX-6P-1-hBRD4-BD1-E151A.

Liu Z., Li Y., Chen H., Lai H.T., Wang P., Wu S.Y., Wold E.A., Leonard P.G., Joseph S., Hu H., Chiang C.M., Brasier A.R., Tian B, & Zhou J. (2022). Discovery, X‑ray Crystallography, and Anti-inflammatory Activity of Bromodomain-containing Protein 4 (BRD4) BD1 Inhibitors Targeting a Distinct New Binding Site. Journal of medicinal chemistry, 65(3), 2388-2408.

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