Pgex kg

Manufactured by GE Healthcare

Sourced in United Kingdom

The PGEX-KG is a protein expression system designed for the production of recombinant proteins in E. coli. It utilizes a strong T7 promoter and a glutathione S-transferase (GST) fusion tag to facilitate high-level expression and purification of target proteins.

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Lab products found in correlation

Histrap ff column, by GE Healthcare (2 mentions) Thrombin protease, by Merck Group (2 mentions) Gst ff column, by GE Healthcare (2 mentions) Dh5α competent cells, by Thermo Fisher Scientific (1 mentions) Gateway cloning technology, by Thermo Fisher Scientific (1 mentions) Pdonr221, by Thermo Fisher Scientific (1 mentions) Glutathione sepharose 4b beads, by GE Healthcare (1 mentions) Pef1 v5 his, by Thermo Fisher Scientific (1 mentions) Pcmv tag2b, by Agilent Technologies (1 mentions) Pegfp n1, by Thermo Fisher Scientific (1 mentions) Pmd18 t, by Takara Bio (1 mentions) Pcdna3.1 his, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

PGEX-KG vectors (3 citations)

8 protocols using pgex kg

Cloning and Purification of CaV2.2 Splice Variants

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For C3_Strep (see: Gardezi et al., 2013 (link)), a PCR fragment of the CaV2.2 long splice (cdB1) variant (aa 2138–2357) was subcloned into pPr-IBA (IBA) expression vector with the Twin-Strep-tag (this was previously named “One-Strep”) at the N-terminus [sequence: SA-WSHPQFEK(GGGS)2GGSAWSHPQFEK (IBA)]. The GST-tagged fusion protein C3_Prox (aa 2138–2299) PCR fragment was subcloned into a pGEX-KG (GE Healthcare) vector and GST-FLAG-tagged proteins (C3_WildF and C3_MutantF, aa 2138–2357) into a pGEX-KG expression vector with a sub-cloned FLAG tag. The DNA sequence in frame was confirmed by sequencing after transformation into DH5α competent cells (Invitrogen). C3_Strep was used on bead whereas GST fusion proteins were eluted using 20 mM reduced Glutathione (Bioshop), in 50 mM Tris-HCl pH 8.0. GST fusion proteins were concentrated using a 10K Microcon and stored in PBS (GIBCO; Life technologies).

Wong F.K., Nath A.R., Chen R.H., Gardezi S.R., Li Q, & Stanley E.F. (2014). Synaptic vesicle tethering and the CaV2.2 distal C-terminal. Frontiers in Cellular Neuroscience, 8, 71.

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Generation of GST-HA-SIK3 and FLAG-HA-SIK3 Constructs

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GFP-HA-SIK3 in pEGFP-C1 was obtained from the University of Dundee (Cat no. DU1338). It was cut with SacI and BamHI and ligated into pGEX-KG (GE Healthcare, Amersham, UK) to generate GST-HA-SIK3^CΔ591 in pGEX-KG. GFP-HA-SIK3 in pEGFP-C1 was cut with BamHI and ligated into BamHI-cut and phosphatase-treated pUHD-P3T/PUR^{28 (link)} to generate FLAG-HA-SIK3 in pUHD-P3T/PUR.

Chen H., Huang S., Han X., Zhang J., Shan C., Tsang Y.H., Ma H.T, & Poon R.Y. (2014). Salt-inducible kinase 3 is a novel mitotic regulator and a target for enhancing antimitotic therapeutic-mediated cell death. Cell Death & Disease, 5(4), e1177-.

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AMPK-α1 Purification and Phosphorylation

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DNA encoding residues 466–525 from human AMPK-α1 were amplified by PCR to include an N-terminal XhoI site and a C-terminal His₆-tag followed by a KpnI site, allowing insertion into pGEXKG (GE Healthcare Life Sciences). Cultures were grown at 37°C until the D₆₀₀ reached ∼0.6, when cultures were induced with 1 mM IPTG and kept at 20°C overnight. The bacteria were pelleted by centrifugation (7500 g, 15 min, 4°C), lysed under liquid N₂ using a pestle and mortar, and resuspended in 50 mM Tris/HCl, pH 8.1, 500 mM NaCl, 20 mM imidazole and the EDTA-free protease inhibitor cocktail (Roche). The protein was purified using a HisTrap FF column (GE Healthcare Life Sciences). Fractions containing protein were pooled and incubated for 30 min at 30°C with 5 mM MgCl₂ and 200 μM ATP-γ-phosphorothioate in the presence or absence of His₆-tagged Akt. The mixture was then applied to a 1 ml GST FF column (GE Healthcare Life Sciences). After washing, the column was loaded with thrombin protease (Sigma) in 50 mM sodium/Hepes, pH 8, and 200 mM NaCl, capped, and left overnight at 4°C. Flow-through fractions containing cleaved phosphorylated or non-phosphorylated peptide was collected.

Hawley S.A., Ross F.A., Gowans G.J., Tibarewal P., Leslie N.R, & Hardie D.G. (2014). Phosphorylation by Akt within the ST loop of AMPK-α1 down-regulates its activation in tumour cells. Biochemical Journal, 459(Pt 2), 275-287.

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Cloning and Expression of Bag101 and Bag102 in S. pombe

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To generate Bag101 (SPBC16G5.11c) constructs, FL (full length), BAG domain (amino acids 78–190) and UBL domain (1–77) cDNAs were amplified from S. pombe genomic DNA and inserted into pDONR221 (Invitrogen) and pGEX-KG (GE Healthcare). Full length bag102⁺ (SPBC530.03c), ΔTM (31–206), BAG (122–206) and UBL (31–121) cDNAs were also inserted into the pDONR221 and pGEX-KG vectors. For expression in S. pombe, the inserts from the pDONR221 vectors were transferred to the pDUAL vector system [54] (link) using Gateway cloning technology (Invitrogen). Both ubp3 variants were cloned via XhoI/NotI into the pJR2-3XL vector with the nmt1⁺ promoter [55] (link). The HA-tagged Spc7-23 construct was obtained by QuikChange site directed mutagenesis (Stratagene) on pJR-XU41 plasmid encoding Spc7-3HA. The expression construct for 6His-ubiquitin has been described before [56] (link). The expression construct for San1 was kindly provided by Dr Makoto Kawamukai [57] (link).

Kriegenburg F., Jakopec V., Poulsen E.G., Nielsen S.V., Roguev A., Krogan N., Gordon C., Fleig U, & Hartmann-Petersen R. (2014). A Chaperone-Assisted Degradation Pathway Targets Kinetochore Proteins to Ensure Genome Stability. PLoS Genetics, 10(1), e1004140.

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Recombinant GST-ALKBH9B Protein Purification

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Full-length atALKBH9B ORF and deletion mutants were subcloned into pGEX-KG (GE Healthcare Life Sciences) to generate a construct with 9B merged to the C-terminal part of the GST. GST and GST:atALKBH9B fusion proteins were expressed in BL21 (DE3) E. coli cells and purified with glutathione Sepharose 4B beads (GE Healthcare Life Sciences) according to the manufacturer’s recommendations. All protein purification procedures were performed at 4°C.

Alvarado-Marchena L., Marquez-Molins J., Martinez-Perez M., Aparicio F, & Pallás V. (2021). Mapping of Functional Subdomains in the atALKBH9B m6A-Demethylase Required for Its Binding to the Viral RNA and to the Coat Protein of Alfalfa Mosaic Virus. Frontiers in Plant Science, 12, 701683.

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AMPK-α1 Phosphorylation Assay

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DNA encoding residues 466-525 from human AMPK-α1 were amplified by PCR to include an N-terminal Xho1 site and a C-terminal (His)₆ tag followed by Kpn1 site, allowing insertion into pGEXKG (GE Life Sciences). Cultures were grown at 37°C until A₆₀₀ ≈ 0.6, when cultures were induced with 1 mM isopropyl β-D-1-thiogalactopyranoside and placed at 20°C overnight. Bacteria were pelleted by centrifugation, lysed under liquid N₂ using a pestle and mortar, and resuspended in 50 mM Tris/HCl, pH 8.1, 500 mM NaCl, 20 mM imidazole with the EDTA-free protease inhibitor cocktail (Roche). The protein was purified using a HisTrap FF column (GE Life Sciences). Fractions containing protein were pooled and incubated for 30 min at 30°C with 5 mM MgCl₂ and 200 μM ATP-γ-phosphorothiate in the presence or absence of (His)₆-tagged Akt. The mixture was then applied to a 1 ml GST FF column (GE Life Sciences). After washing, the column was loaded with thrombin protease (Sigma) in 50 mM Na Hepes, pH 8, 200 mM NaCl, capped, and left overnight at 4°C. Flow-through fractions containing cleaved phosphorylated or non-phosphorylated peptide was collected.

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Expression Constructs for Mouse Siglec-G-ITIM and Associated Proteins

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To generate a construct expressing mouse Siglec-G-ITIM, cDNA for Siglec-G-ITIM was amplified by RT-PCR with the primers (5'-AGGCAGA TCTCAG AAGAAAGGAACCCAGGAGG-3', 5'-AAGCTTGTGGACT CTGACCTCTGTGTA-3') and subcloned into expression vector pCMVTag2B (Agilent, Santa Clara, CA), yielding the plasmid pCMV-Tag2B-Flag-ITIM. To generate a construct expressing mouse Rab1a, cDNA for Rab1a was amplified by RT-PCR with the primers (5'-AGTGACGGATCCGCCACCATGTCCAGCATGA A TCCCG-3', 5'-CAGATTCTCGAGCCAGCAGCCTCCAC CTGAC-3') and subcloned into expression vector pCDNA6/myc-His (Life Technologies, Carlsbad, CA), yielding the plasmid pCDNA6/myc-His-Rab1a. To generate a construct expressing mouse Gdi2, cDNA for Gdi2 was amplified by RT-PCR with the primers (5'-AGGATCCGCCACCATGAATGAGGAATAC GACGT-3', 5'-CGAATTCTCCATAAATGTCAT-3') and subcloned into expression vector pEF1/V5-His (Life Technologies), yielding the plasmid pEF1/V5-His-Gdi2. To generate a construct expressing mouse Siglec-G-ITIM-GST, cDNA for Siglec-G-ITIM was amplified by RT-PCR with the primers (5'-AGATCTCAGAAGAAAGGAACCCAGGAGGAAC-3', 5'-AAGCTTGTGGACTCTGACCTCTGTGTAAT-3') and subcloned into expression vector pGEX-KG (GE Healthcare, Chicago, IL), yielding the plasmid pGEX-KG-ITIM. Underlined nucleotides were added for restriction enzyme digestion. All constructs were verified by restriction enzyme digestion and DNA sequencing.

Wu Y., Yang D, & Chen G.Y. (2022). The role of the Siglec-G ITIM domain during bacterial infection. Cellular and molecular biology (Noisy-le-Grand, France), 67(4).

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Optimizing mHCN1 Construct Cloning

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Four vectors, pMD18-T (Takara Bio Inc., Otsu, Japan), peGFP-N1 (Invitrogen, Carlsbad, CA, USA), pGEX-KG (GE Healthcare, Buckinghamshire, UK), and pcDNA3.1/His (Invitrogen), were used in this study. The cDNA of mouse hyperpolarization-activated cyclic nucleotide-gated action channel1 (mHCN1; NM_010408, 2733 bp) was used as the insert template. The primers were synthesized by Genewiz (Genewiz Biotechnology Suzhou, China).
Several primers were designed to generate a series of truncated mHCN1 PCR products (114-2787 bp) to determine whether the insertion size would influence the rate of recombination. All primers consisted of a 27-nucleotide homologous arm at the 5'-end, and a segment complimentary to the template DNA at the 3'-end. All primers utilized in this study are listed in Table 1.

Wang Y., Liu Y., Chen J., Tang M.J., Zhang S.L., Wei L.N., Li C.H, & Wei D.B. (2015). Restriction-ligation-free (RLF) cloning: a high-throughput cloning method by in vivo homologous recombination of PCR products. Genetics and molecular research : GMR, 14(4).

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