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Pgex4t2 expression vector

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The PGEX4T2 expression vector is a laboratory tool used for the expression and purification of recombinant proteins in Escherichia coli (E. coli) cells. It contains the glutathione S-transferase (GST) gene, which allows for the fusion of the target protein to the GST tag, facilitating affinity-based purification of the expressed protein.

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Lab products found in correlation

Ni nta resin, by Qiagen (1 mentions) Anti gst antibody, by GE Healthcare (1 mentions) Bca protein assay kit, by Thermo Fisher Scientific (1 mentions) Glutathione sepharose 4b beads, by GE Healthcare (1 mentions) T4 dna ligase, by New England Biolabs (1 mentions) Q5 hot start master mix, by New England Biolabs (1 mentions) M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions)

5 protocols using pgex4t2 expression vector

1

Recombinant Protein Production and Purification

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The coding sequences of the B3 domain of rice OsGD1 (Os07g0563300, amino acids 342–640) and OsVAL2 (Os07g0679700, amino acids 144–517) were amplified from rice cDNA and subcloned into the pGoldI expression vector (TAKARA, Japan) to generate the 6× his-tagged recombinant rOsGD1-B3 and rOsVAL2-B3 proteins. The recombinant proteins were extracted from Escherichia coli BL21 cells and purified by Ni-NTA resin (Qiagen, Germany) according to the manufacturer’s protocol. The MADS3 (Os01g0201700) coding sequence was subcloned into the pGEX4T-2 expression vector (GE Healthcare, USA) to generate the GST-tagged rOsMADS3 proteins. The rOsMADS3 protein was extracted from E. coli BL21 cells and purified by GSH-agarose (GE Healthcare, USA) according to the manufacturer’s protocol. To test the protein quality, the proteins were separated in SDS-PAGE gel and visualized by Coomassie Brilliant Blue staining and western blotting with corresponding antibodies (Figure S2).
Xie Y., Zhang Y., Zhao X., Liu Y.G, & Chen L. (2016). A CAPS-based binding assay provides semi-quantitative validation of protein-DNA interactions. Scientific Reports, 6, 21030.
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2

Recombinant ANISERP Protein Expression and Purification

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The AniSerp gene without the sequence coding for the predicted putative N-terminal signal peptide was subcloned into the pGEX4T2 expression vector (GE Healthcare Life Sciences) and further transformed into the BL21 strain of E. coli (F-, ompT, hsdS [rb-, mb-], gal) (GE Healthcare Life Sciences). Recombinant protein expression was induced by different protocols to obtain a soluble fusion protein similar to the native protein, which was subsequently purified. Expression protocols including different combinations of incubation temperatures (16 and 37 °C), concentrations of isopropyl β-D-1-thiogalactopyranoside (ITPG) (0.01, 0.1, 0.5 and 1 mM) and incubation times (4-16 h) were used. Once the recombinant ANISERP protein was obtained in its soluble form, it was purified by glutathione-Sepharose 4B bead affinity chromatography and eluted with 15 mM glutathione, 100 mMTris-HCl pH 8, 0.1 % Triton X-100 elution buffer. The purified recombinant protein was then dialyzed. The protein concentration was determined using the BCA Protein Assay Kit (Thermo Scientific), with bovine serum albumin (BSA) as a standard [22 (link)]. The expressed protein was then subjected to SDS-PAGE analysis and Coomassie staining, plus Western blot analysis with an anti-GST antibody (GE Healthcare Life Sciences).
Valdivieso E., Perteguer M.J., Hurtado C., Campioli P., Rodríguez E., Saborido A., Martínez-Sernández V., Gómez-Puertas P., Ubeira F.M, & Gárate T. (2015). ANISERP: a new serpin from the parasite Anisakis simplex. Parasites & Vectors, 8, 399.
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3

Recombinant Expression and Purification of Bhlh-tun1

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The nucleotide sequence encoding the entire Bhlh-tun1 protein was amplified from Ciona cDNA using primers
(restriction sites are underlined):
bHLH1-CDS-5: 5’-gccagaattcGCAAAAATGGTTAAAGCGAGCCCGATC-3’
bHLH1-CDS-3: 5’-tccactcgagGATTCACTCTCGCGTTCTGGAATTG-3’
and cloned in-frame with a GST tag into the XhoI/EcoRI sites of the pGEX-4T-2 expression vector (GE Healthcare,
Piscataway, NJ). The resulting plasmid was transformed into chemically competent BL21 (DE3) E. coli. Bacteria
were grown at 25°C, and protein expression was induced by adding 0.5 mM IPTG to the medium for 3 hrs. The GST-Bhlh-tun1
fusion protein was purified from crude bacterial extracts using Glutathione Sepharose 4B beads (GE Healthcare, Piscataway, NJ)
according to the manufacturer’s instructions, and eluted in 50 mM Tris pH 8.0 containing 10 mM reduced glutathione.
Kugler J.E., Wu Y., Katikala L., Passamaneck Y.J., Addy J., Caballero N., Oda-Ishii I., Maguire J.E., Li R, & Di Gregorio A. (2019). Positioning a multifunctional basic helix-loop-helix transcription factor within the Ciona notochord gene regulatory network. Developmental biology, 448(2), 119-135.
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4

Construction of GST-VlsE Fusion Protein

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To generate an expression plasmid encoding an N-terminal GST fusion of the VlsE B31 A3 protein, genomic DNA was prepared from B. burgdorferi strain B31 A3 [28 (link)]. The full length vlsE gene (lacking the putative lipoprotein signal sequence) was PCR amplified, with restriction sites added using Q5 Hot Start Master Mix (New England Biolabs) and the primers prMP131 and prMP132. Primer sequences are detailed in S1 Table. The PCR fragment was then inserted into the BamHI and SalI sites of pGEX4T2 expression vector (GE Healthcare Life Sciences) using T4 DNA Ligase (New England Biolabs). To generate the VlsEECM mutant, residues at positions 163, 167, and 170 of B. burgdorferi B31 A3 VlsE were first changed to methionine residues using overlap extension PCR and primers prMP138 and prMP139. Primers prMP144 and prMP145 were then used to generate the final K>M substitution at position 159. All ligation reactions were directly electroporated into E. coli DH5α. Carbenicillin resistant colonies were screened by BamHI and SalI restriction digest and gel electrophoresis on a 1% agarose gel run for one hour at 75 V. Clones containing the correct insert size were subjected to Sanger sequencing. The cloning of the pGEX-4T2 plasmid encoding ospD from B. burgdorferi strain B31 has been described previously [37 (link)]. Confirmed plasmids were subsequently used to transform E. coli BL21(DE3).
Tan X., Lin Y.P., Pereira M.J., Castellanos M., Hahn B.L., Anderson P., Coburn J., Leong J.M, & Chaconas G. (2022). VlsE, the nexus for antigenic variation of the Lyme disease spirochete, also mediates early bacterial attachment to the host microvasculature under shear force. PLoS Pathogens, 18(5), e1010511.
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5

Measuring Active RhoA Levels

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RhoA. The RhoA-binding domain (RBD) of the rhotekin protein was amplified by PCR from the MDA-MB-231 cell line cDNA bank (contained in our laboratory at the International Joint Cancer Institute, Second Military Medical University), cloned into the pGEX-4T-2 expression vector (Amersham; GE Healthcare, Chicago, IL, USA), and was expressed as a glutathione S-transferase (GST) fusion protein in BL21 (DE3) cells (International Joint Cancer Institute, Second Military Medical University). The levels of GTP-bound RhoA (active RhoA) in cell lysates were measured using immunoprecipitation, as previously described (23) . Briefly, control and EphA3-overexpressing KYSE510 cells were lysed in M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Inc.) containing protease inhibitor cocktail (Roche Diagnostics) and were precleared with glutathione beads. The GST-RBD protein was allowed to bind to the glutathione bead at 4˚C overnight and the supernatants of the precleared protein lysates were applied to these beads, allowing the active form of RhoA to bind to the RBD domain. Total RhoA levels in the cell lysate were compared to the pulled-down active RhoA by western blotting and were measured using Bio-Rad Quantity One 4.52 software (Bio-Rad Laboratories, Inc.).
Chen X., Lu B., Ma Q., Ji C.D, & Li J.Z. (2019). EphA3 inhibits migration and invasion of esophageal cancer cells by activating the mesenchymal‑epithelial transition process. International journal of oncology, 54(2).
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