Pgex2tk

Manufactured by GE Healthcare

The PGEX2TK is a laboratory equipment product from GE Healthcare. It is designed for gene expression and protein purification. The core function of the PGEX2TK is to facilitate the expression and purification of recombinant proteins in Escherichia coli.

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Lab products found in correlation

Pgex 5x 1, by GE Healthcare (1 mentions) Pgex 6p 1, by GE Healthcare (1 mentions) Glutathione resin, by GE Healthcare (1 mentions) Phusion polymerase, by New England Biolabs (1 mentions) Sema3a, by Thermo Fisher Scientific (1 mentions) Slit2n, by Thermo Fisher Scientific (1 mentions)

4 protocols using pgex2tk

Recombinant Protein Production Protocols

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Experimental procedures are described in greater detail in the Supplemental Material.
Briefly, the UBA domains of CBLB (aa 914–982) and Cbl (aa 839–906)
were cloned into pGEX 2TK or pGEX 5X-1 (GE Healthcare Biosciences, Pittsburgh,
PA), respectively, as previously described.^{12 (link)} The N-terminal regions containing the tyrosine-binding
domain, linker, and RF of CBLB (aa 2–448) and Cbl (aa 2–469) were
cloned into pGEX 6P-1 as previously described.^{4 (link),13 (link)}The bacterial expression construct for Ube2d2, pETb-UbcH5b, was kindly provided
by Dr. Allan Weissman. Recombinant GST proteins were produced as previously
described.^{13 (link)}Recombinant Ube2d2 was produced and purified as previously described.^{14 (link)} Recombinant HRD1
(HMG-CoA reductase
degradation 1) protein was kindly provided by Allan
Weissman.
Large-scale preparation of recombinant proteins was performed by the
Protein Expression Laboratory, Frederick National Laboratory for Cancer
Research, by modification of the above procedures.

Wilson B.A., Voeller D., Smith E.A., Wamiru A., Goncharova E.I., Liu G., Lipkowitz S, & O’Keefe B.R. (2021). In Vitro Ubiquitination Platform Identifies Methyl Ellipticiniums as Ubiquitin Ligase Inhibitors. SLAS discovery : advancing life sciences R & D, 26(7), 870-884.

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Recombinant M2eH-A-S-H Protein Production

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Synthetic M2eH-A-S-H ORF was digested with BamHI and EcoRI enzymes and cloned into a commercial vector, pGEX2TK (GE Healthcare). The resulting plasmid, pM2eH-A-S-H-GST, was verified by restriction analysis and nucleotide sequencing. pM2eH-A-S-H-GST was used to transform BL21 Escherichia coli strain, and the recombinant strain was used to overproduce M2eH-A-S-H-GST after addition of IPTG (final concentration—1 mM). The protein was purified by affinity chromatography on glutathione resin (GE Healthcare). 0.2 mg of pure M2eH-A-S-H-GST protein was obtained from 0.25 L culture.

Łęga T., Weiher P., Obuchowski M, & Nidzworski D. (2016). Presenting Influenza A M2e Antigen on Recombinant Spores of Bacillus subtilis. PLoS ONE, 11(11), e0167225.

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Recombinant Expression of Guidance Cues

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Met-Robo cDNA was kindly provided by Elke Stein (Yale University, New Haven, CT) (Stein and Tessier-Lavigne, 2001 (link)). RoboCC domains were amplified from Met-Robo cDNA using Phusion polymerase (New England Biolabs, Inc.). Amplified products were inserted into pIC113 (Cheeseman and Desai, 2005 (link); gift from I. Cheeseman, Massachusetts Institute of Technology, Cambridge, MA) between the EcoRI-SalI sites. Mena EVH1 (amino acids 1–115) was amplified by PCR from mouse cDNA and inserted into pGEX2TK (GE Healthcare). Recombinant GST-EVH1 protein was produced in BL-21 Escherichia coli and purified using standard methods. Slit2N and Sema3A were purchased from PeproTech.

McConnell R.E., Edward van Veen J., Vidaki M., Kwiatkowski A.V., Meyer A.S, & Gertler F.B. (2016). A requirement for filopodia extension toward Slit during Robo-mediated axon repulsion. The Journal of Cell Biology, 213(2), 261-274.

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Construction and Expression of Recombinant Proteins

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The construction of bacterial expression vectors containing the cDNA sequences of human Ets-1 p51, Ets-1 p27 and N-terminal deletion mutant ΔN331 have been previously described [17, 18] . The construction of pTyb2 vector expressing human Ets-1 p51 fused to a biotin tag has been previously described [8] . pGEX-2TK vectors expressing human full-length Ku70 and Ku80 fused to a GST as well as deletion mutant constructions of GST-Ku70 were kindly provided by Professor S. P. Jackson (University of Cambridge, UK) [19] . pET32a-His-PARP-1 24 and pET32a-His-PARP-1 89 vectors, expressing the caspase cleavage products of PARP-1 with hexahistidine tag (His), were generously provided by Professor M. S. Satoh (Laval University, Canada) [20] . To produce BRCT domain of PARP-1 fused to a GST tag, the sequence coding amino acids 378-479 of PARP-1 was obtained by PCR amplification, using the following primers: 5ʹ-CAGT AGGGATCCGCTGCTGTGAACTCCTCTGC-3ʹ and 5ʹ-GTCAACGAATTCGGACAAGATGTGCGCTAAG A-3ʹ. The PCR product was then cloned between the BamHI and EcoRI restriction sites in a pGEX-2TK (GE Healthcare®) prokaryotic expression vector. Constructs were checked for nucleotide sequence.

Choul-Li S., Legrand A.J., Bidon B., Vicogne D., Villeret V, & Aumercier M. (2018). Ets-1 interacts through a similar binding interface with Ku70 and Poly (ADP-Ribose) Polymerase-1. Bioscience, biotechnology, and biochemistry, 82(10).

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