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Pgex 4t 2

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The PGEX-4T-2 is a laboratory equipment product offered by GE Healthcare. It is a plasmid vector designed for the expression of recombinant proteins in Escherichia coli. The plasmid features a tac promoter, which allows for the inducible expression of the target protein.

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Lab products found in correlation

Glutathione sepharose 4b, by GE Healthcare (4 mentions) T4 dna ligase, by Thermo Fisher Scientific (4 mentions) Glutathione agarose beads, by Thermo Fisher Scientific (3 mentions) Pegfp c1, by Takara Bio (3 mentions) Pcmv myc, by Takara Bio (2 mentions) Glutathione chromatography, by BD (2 mentions) Pgex 4t 2 vector, by Takara Bio (2 mentions) Flag l3mbtl2, by Addgene (2 mentions) Flag hmgb1 plasmids, by Addgene (2 mentions) Flag l3mbtl2, by Takara Bio (2 mentions) Lenticrispr v2, by Addgene (2 mentions) Pgex 4t 1, by GE Healthcare (2 mentions) High fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions) Anti c myc 9e10, by Santa Cruz Biotechnology (1 mentions) Pcmv flag, by Takara Bio (1 mentions) Anti myc, by Takara Bio (1 mentions) Anti actin, by Merck Group (1 mentions) Anti ha, by Roche (1 mentions) Pcmv ha, by Takara Bio (1 mentions) Anti gfp, by Santa Cruz Biotechnology (1 mentions)

Close spelling variants of this product

PGEX-4T-2 vector (13 citations) PGEX-4T (7 citations)

52 protocols using pgex 4t 2

1

Cloning and Expression of CDC33 (eIF4E) from S. cerevisiae

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The CDC33 (eIF4E) gene was amplified from S. cerevisiae genomic DNA using Pfu DNA polymerase (Fermentas) with eIF4Ef and eIF4Er primers containing NcoI and HindIII restriction sites as follows: 5 min at 95°C; 25 cycles of 30 s at 94°C, 30 s at 55°C, and 2 min at 72°C; and finally, 10 min at 72°C. The PCR cassette was digested and inserted into the NcoI and HindIII restriction sites of pGEX-4T2 (GE Healthcare) to generate a pGEX4T2::CDC33 construct. A pYX212 yeast shuttle plasmid (Ingenius) was digested with EcoRI and XhoI restriction endonucleases. A sequence encoding the K1 toxin was excised from a pYX213::M1 (Valis et al., 2006 (link)) plasmid using the same restriction enzymes and ligated into the digested pYX212 plasmid to generate the plasmid pYX212::M1. All clones were verified by restriction endonuclease digestion and sequencing. All primers and plasmids used in this study are listed in Supplementary Tables S1, S3, respectively.
Vopálenský V., Sýkora M., Mašek T, & Pospíšek M. (2019). Messenger RNAs of Yeast Virus-Like Elements Contain Non-templated 5′ Poly(A) Leaders, and Their Expression Is Independent of eIF4E and Pab1. Frontiers in Microbiology, 10, 2366.
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2

Cloning and Mutagenesis of DNA Repair Proteins

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Full length cDNA of hAPE1 was amplified by PCR using pET28-hAPE1 plasmid (a gift from Dr. Alex Drohat, University of Maryland Medical School) as template and the primers listed in Additional file 1: Table S1. The PCR product was digested with BamHI and XhoI and ligated into the digested pGEX-4T-2 (GE Healthcare) to yield the pGEX-hAPE1 construct.
Plasmids containing GST-hMYH, GST-hMYH(1–315), GST-hMYH(1–350), GST-hMYH(1–350)V315A, GST-Rad9, GST-Rad1, and GST-Hus1 have been described by Shi et al. [13 (link)]. The plasmid containing GST-hMYH(65–350) was derived from pET19b-hMYH(65–350) [11 (link)] by PCR amplification using primers listed in Additional file 1: Table S1. The PCR product was digested with BamHI and XhoI and ligated into the digested pGEX-4T-2 (GE Healthcare). The Gln324 to His (Q324H) mutant of the hMYH gene was constructed with the QuickChange site-directed mutagenesis kit (Stratagene) using the pGEX4T-hMYH(1–350) plasmid [13 (link)] as template and primers listed in Additional file 1: Table S1. The mutation was verified by DNA sequencing.
Hwang B.J., Jin J., Gao Y., Shi G., Madabushi A., Yan A., Guan X., Zalzman M., Nakajima S., Lan L, & Lu A.L. (2015). SIRT6 protein deacetylase interacts with MYH DNA glycosylase, APE1 endonuclease, and Rad9–Rad1–Hus1 checkpoint clamp. BMC Molecular Biology, 16, 12.
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3

Recombinant GST-tagged OspC Proteins

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To generate recombinant glutathione-S-transferase (GST)-tagged OspC proteins, the ospC open reading frames lacking the putative signal sequences from B. burgdorferi strains B31-A3 and N40 clone D10/E9, and B. garinii strain PBr were amplified and cloned in pGEX4T2 (GE Healthcare, Piscataway, NJ) as previously described (Benoit, Fischer, Lin, Parveen, & Leong, 2011 (link)) (Table S4). Amplified fragments were engineered to encode a BamHI site at the 5’ end and a stop codon followed by a SalI site at the 3’ end. PCR products were sequentially digested with BamHI and SalI and then inserted into the BamHI and SalI sites of pGEX4T2 (GE Healthcare, Piscataway, NJ). The resulting plasmids were transformed into E. coli strain BL21(DE3) (Table S1) and induced for production of GST-tagged OspC variants. Proteins were purified by glutathione chromatography according to the manufacturer’s instructions (BD Biosciences, San Jose, CA). Antisera against OspCB31-A3, OspCN40-D10/E9, and OspCPBr were generated by immunizing five-week-old BALB/C mice with each of the OspC proteins as described previously (Barthold, Hodzic, Tunev, & Feng, 2006 (link)).
Caine J.A., Lin Y.P., Kessler J.R., Sato H., Leong J.M, & Coburn J. (2017). Borrelia burgdorferi outer surface protein C (OspC) binds complement component C4b and confers bloodstream survival. Cellular microbiology, 19(12), 10.1111/cmi.12786.
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4

Recombinant Expression of TIA-1 RRM2,3 Protein

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To construct the plasmid for expression of TIA-1 RRM2,3 (93-274), pGEX-4T-2 (GE Healthcare) was modified to bear a 6xHis tag and a thrombin cleavage site. TIA-1 RRM2,3 was inserted directly after the thrombin site using oligonucleotides TIA-1 RRM2,3-For (GCG​TGG​ATC​CCC​AGG​AAT​TCC​CAA​TCA​TTT​CCA​TGT​CTT​TGT​TGG​TG) and TIA-1 RRM2,3-rev (CAG​TCA​CGA​TGC​GGC​CGC​TTA​TTT​GCC​CCA​ATA​GCA​TTT​CAC​AAC). To construct the plasmid TIA-1 RRM2,3 bearing mutations H94A, H96 A and H94A H96A, site-directed mutagenesis by inverse PCR (Liu and Naismith, 2008 (link)) was performed using the oligonucleotide pairs:
TIA-1 RRM2,3-H94A-For: CCA​ATg​ctT​TCC​ATG​TCT​TTG​TTG​GTG​ATC​TCA​GCC​CAG
TIA-1 RRM2,3-H94A-Rev: ATG​GAA​agc​ATT​GGG​AAT​TCC​TGG​GGA​TCC​ACG
TIA-1 RRM2,3-H96A-For: TTT​Cgc​tGT​CTT​TGT​TGG​TGA​TCT​CAG​CCC​AG
TIA-1 RRM2,3-H96A-Rev: AAA​GAC​agc​GAA​ATG​ATT​GGG​AAT​TCC​TGG​GGA​TCC​ACG
TIA-1 RRM2,3-H94A H96A-For: CCA​ATg​ctT​TCg​cTG​TCT​TTG​TTG​GTG​ATC​TCA​GCC​CAG
TIA-1 RRM2,3-H94A H96A-Rev: AAA​GAC​agc​GAA​agc​ATT​GGG​AAT​TCC​TGG​GGA​TCC​ACG​CGG
West D.L., Loughlin F.E., Rivero-Rodríguez F., Vankadari N., Velázquez-Cruz A., Corrales-Guerrero L., Díaz-Moreno I, & Wilce J.A. (2022). Regulation of TIA-1 Condensates: Zn2+ and RGG Motifs Promote Nucleic Acid Driven LLPS and Inhibit Irreversible Aggregation. Frontiers in Molecular Biosciences, 9, 960806.
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5

Protein-Protein Interaction Assay

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To construct GST-fusion plasmids, Avr3b was inserted into the vector pGEX4T-2 (GE Healthcare Life Science). To construct His-fusion plasmid, GmCYP1 was inserted into the vector pET28a. Pull-down assay was performed using ProFound pull-down GST protein-protein interaction kit (Pierce) according to the manufacturer’s instructions. GST, GST-Avr3b, and His-GmCYP1 was expressed in E. coli strain BL21 (DE3) respectively. The soluble total proteins were incubated with 50 μl glutathione agarose beads (Invitrogen) for 2 hours at 4°C. The beads were washed five times and then incubated with equal amount of bacterial lysates containing His-GmCYP1 for another hour at 4°C. The beads were washed five times again, and the presence of His-GmCYP1 was detected by western blot using anti-His antibody.
Kong G., Zhao Y., Jing M., Huang J., Yang J., Xia Y., Kong L., Ye W., Xiong Q., Qiao Y., Dong S., Ma W, & Wang Y. (2015). The Activation of Phytophthora Effector Avr3b by Plant Cyclophilin is Required for the Nudix Hydrolase Activity of Avr3b. PLoS Pathogens, 11(8), e1005139.
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6

Cloning and Expression of PvPHIST/CVC-81 Fragments

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Two fragments of pvphist/cvc-8195 (PlasmoDB no. PVX_093680) were amplified by PCR from cDNA as described previously [18 (link)]. Fragment PvPHIST/CVC-8195-NT comprised amino acid residues 1–170 of the full-length PvPHIST/CVC-8195 amino acid sequence (forward primer 5′-gatccccaggaattcccATGAGTCCCTGCAACATCC-3′ and reverse primer 5′-atgcggccgctcgagTTAAGCTGGTTGATCGGGCCTA-3′). Fragment PvPHIST/CVC-8195-CT comprised residues 556–710 (forward primer 5′-gatccccaggaattcccGACAATGAACAACTCCCATTCG-3′ and reverse primer 5′-atgcggccgctcgagTTAGAGTTTGCTGTGTTTCTTCATCT-3′). The underline and lowercase letter of primer sequence indicate homologous sequence to the vector sequence and restriction enzyme site, respectively. The PCR amplification products with high-fidelity DNA polymerase (Finnzymes, Espoo, Finland) were ligated into the expression vector pGEX 4T-2 (GE Healthcare Life Sciences, Uppsala, Sweden). Positive clones were validated by DNA sequencing analysis. The correct clones were then transformed into competent BL21 StarTM (DE3) cells of E. coli (Invitrogen, Seoul, Korea) for protein expression. Soluble protein was purified on Glutathione SepharoseTM 4B (GE Healthcare Life Sciences) columns according to the manufacturer’s protocol.
Wang B., Lu F., Han J.H., Lee S.K., Cheng Y., Nyunt M.H., Ha K.S., Hong S.H., Park W.S, & Han E.T. (2016). Characterization of Caveola-Vesicle Complexes (CVCs) Protein, PHIST/CVC-8195 in Plasmodium vivax. The Korean Journal of Parasitology, 54(6), 725-732.
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7

Plasmid Construction and Antibody Details

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RLIM and c-Myc expression plasmids were constructed by cloning human RLIM (NM_016120) and c-MYC (NM_002467) ORF into pCMV-HA (Clontech) and pCMV-myc (Clontech) vectors respectively. RLIMC596A and c-MycT58A/S62A expression plasmids were constructed by target point mutagenesis (Strategene). Human ubiquitin ORF were cloned into pCMV-flag (Clontech) and pcDNA3.1-his (ThermoScientific) vectors respectively. For bacterial expression, RLIM and c-MYC ORF were cloned into pET28a (6×His) (Clontech) and pGEX-4T-2 (GE Healthcare Life Sciences) vectors respectively.
The antibodies used were anti-RLIM (M01, Abnova, 1:1000), anti-c-Myc (9E10, Santa Cruz, 1:200), anti-HA (Roche, 1:1000), anti-myc (Clontech, 1:1000), anti-Flag (M2, Sigma, 1:1000), anti-actin (Sigma, 1:10000) and anti-GFP (Santa Cruz, 1:1000).
Gao R., Wang L., Cai H., Zhu J, & Yu L. (2016). E3 Ubiquitin Ligase RLIM Negatively Regulates c-Myc Transcriptional Activity and Restrains Cell Proliferation. PLoS ONE, 11(9), e0164086.
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8

Recombinant Expression of Skap2 and WASp Domains

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Skap2 and WASp cDNA was obtained by reverse transcription PCR strategy from mouse neutrophil RNA. The Skap2-CC domain (40–122 aa), -PH domain (301–666 aa), and -SH3 domain (886–1,077 aa) fragments, truncated Skap2 lacking the SH3 domain (Skap2ΔSH3; 1–885 aa), and truncated WASp lacking the VCA domain (WASpΔVCA; 1–1,226 aa) were generated by PCR-based techniques. Skap2 mutations R140M and W336K were generated by QuickChange site-directed mutagenesis (Agilent Technologies). For expression of fusion proteins, cDNA was subcloned into pGEX-4T-2 (GE Healthcare) or pET30a (EMD Millipore). The cytoplasmic domain of CD18 cloned into pGEX-4T-1 was provided by S. Kliche (University of Magdeburg, Magdeburg, Germany). To generate recombinant retrovirus, cDNAs were subcloned in pMSCV-IRES-GFP II (pMIG II).
Boras M., Volmering S., Bokemeyer A., Rossaint J., Block H., Bardel B., Van Marck V., Heitplatz B., Kliche S., Reinhold A., Lowell C, & Zarbock A. (2017). Skap2 is required for β2 integrin–mediated neutrophil recruitment and functions. The Journal of Experimental Medicine, 214(3), 851-874.
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9

Construction of AID and V5-PK Tagging Plasmids

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Auxin-inducible degron (AID) tagging was performed using a plasmid, DHB137, which we constructed by inserting three HA epitopes in fusion with the three copies of the mini-AID sequence from pMK15156 (link). V5-PK tagging was performed using a plasmid, DHB123, which we constructed by inserting three V5 epitopes 5′ to the hphMX6 cassette into pFA6a-hphMX6 (Euroscarf #P30438)51 (link). For GST pull-down assays, DNA fragments comprising either nucleotides +925 to +1054 from the S. pombe spt20 ORF, encoding residues Asp282 to Ala324, or nucleotides +1402 to +1611 from the S. cerevisiae SPT20 ORF, encoding residues Met468 to Ala537, were synthesised and amplified. Each product was then subcloned into pGEX-4T2 (GE Healthcare Life Sciences), 3′ and in frame to the GST coding sequence, using the Gibson assembly kit (E2611L, New England Biolabs), to generate the DHB179 and DHB193 plasmids, respectively.
Elías-Villalobos A., Toullec D., Faux C., Séveno M, & Helmlinger D. (2019). Chaperone-mediated ordered assembly of the SAGA and NuA4 transcription co-activator complexes in yeast. Nature Communications, 10, 5237.
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10

Purification of GST-tagged avHaCE

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WT HaCE (avi_3097) containing GST at the N-terminus, was generated by QuikChange site-directed mutagenesis using a GST-tagged avGGAAF construct in vector pGEX-4T-2 (GE Healthcare) as the template. GST-tagged avHaCE was grown in Lennox L Broth (10 grams tryptone, 5 grams sodium chloride, 5 grams yeast extract per liter) to an OD600 of ~0.6, after which expression was induced by the addition of 250 μM IPTG and allowed to proceed for 16 hours at 18 °C under agitation (250 rpm). Cells were harvested by centrifugation and then lysed in phosphate buffered saline (PBS, 8 g NaCl, 2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4 per liter, pH 7.4) containing 1% Triton X-100 and 1 mM PMSF. Cellular debris were removed as described above and the cleared lysate was loaded onto Glutathione Sepharose 4B resin and washed with 10 column volumes of PBS. GST-tagged avHaCE was eluted from the column using 50 mM Tris-HCl (pH 8.0) containing 20 mM reduced L-glutathione. Fractions eluted from the column were analyzed by SDS-PAGE. The fractions containing the fused protein were pooled and concentrated, and then desalted, to remove glutathione, into 50 mM Tris-HCl (pH 8.0) buffer using a PD-10 column.
Williams D.E., Nesbitt N.M., Muralidharan S., Hossain S, & Boon E.M. (2023). H-NOX Regulates Biofilm Formation in Agrobacterium Vitis in Response to NO. Biochemistry, 62(4), 912-922.
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