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Pgex 4t 1 expression vector

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The PGEX-4T-1 expression vector is a plasmid designed for the expression of recombinant proteins in Escherichia coli. It contains the tac promoter for inducible expression and the glutathione S-transferase (GST) gene for affinity purification of the expressed proteins.

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Lab products found in correlation

E coli top10 competent cells, by Thermo Fisher Scientific (1 mentions) Genelute plasmid miniprep kit, by Merck Group (1 mentions) Desalting column, by GE Healthcare (1 mentions) Glutathione sepharose 4b column, by GE Healthcare (1 mentions) One shot bl21 star de3, by Thermo Fisher Scientific (1 mentions) Glutathione sepharose 4b, by GE Healthcare (1 mentions) Akta fplc system, by GE Healthcare (1 mentions) Edta free protease inhibitor cocktail, by Roche (1 mentions) Superdex 75 fplc column, by GE Healthcare (1 mentions) Hepes, by Merck Group (1 mentions) Sodium chloride (nacl), by Merck Group (1 mentions) Gst binding buffer, by Sangon (1 mentions) Anti his antibody and anti gst antibody, by Abmart (1 mentions) Gst sefinose kit, by Sangon (1 mentions) Gst resin, by Sangon (1 mentions) Pet 29b expression vector, by Merck Group (1 mentions) His bind purification kit, by Merck Group (1 mentions) Pet sumo vector, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

PGEX-4T-1 (217 citations) PGEX-4T-1 vector (106 citations) PGEX-4T vector (22 citations) PGEX vector (21 citations) PGEX-4T (7 citations) PGEX-4T expression vector (2 citations)

12 protocols using pgex 4t 1 expression vector

1

Functionalization of PEG Hydrogels for Cell Adhesion

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Example 9

In order to seed cells on the HAHL-device, the otherwise inert PEG hydrogel needs to be rendered cell-adherent. A recombinant fibronectin domain 9 and 10 domain was used to promote cell adhesion. The corresponding synthetic gene was ordered from Eurofins™ (France). Cloning in pGEX-4T-1 expression vector (GE Lifesciences), expression and purification was performed at the Platform of recombinant proteins at Institut Pasteur. The FN9-10 fragment was expressed in E. coli BL21 Star™ (DE3) in 4L cultures. Purification of the recombinant protein was performed on GST columns followed by a thrombin cleavage of the purification tag. Purified FN9-10 fragment was concentrated at 3.5 mg·ml−1 in PBS. the resultant FN9-10 fragment was then coupled, via its free secondary amines, to a 3.5 kDa heterofunctional NHS-PEG-Maleimide linker (NOF Corporation) with a stoichiometric ratio of 1:4. Incubation was carried out for 1 hour at room temperature 500 μl of FN9-10 at 3.5 mg·ml−1 with 29 μl PEG linker at 50 mg·ml−1. PEGylated FN9-10 solution was then perfused in the microfluidic channel and on the free surface of the hydrogel. Treated HAHL-device was then incubated for 2 hours at 37° C. Finally, the excess of FN9-10 was washed away with a PBS wash.

US11643625B2. Hydrogel-based organ-on-chip microfluidic device (2023-05-09). INSTITUT PASTEUR [FR]. Inventors: Samy Olivier Gobaa [FR].
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2

Cloning and Characterization of Crotalus adamanteus Disintegrin

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A full-length cDNA encoding a Crotalus adamanteus venom metalloproteinase II was used (GenBank accession no. JX457344) as a PCR template to subclone its disintegrin domain. PCR was used to generate double stranded cDNA, with the following disintegrin-specific primers (a forward primer 5′-CGCGAATTCGAGGTGGGAGAAGATTGTGACTG-3′ and a reverse primer 5′-GACTCGAGTTAGCCATAGAGGCCATTTCTGGGA-3′, two restriction enzyme sites (underlined): EcoRI in forward primer and XhoI in reverse primer) as previously described (Suntravat et al, 2013 (link)). PCR amplification consisted of a cycle of 94°C (3min), 40 cycles of 94°C (30sec), 60°C (30sec), and 72°C (1min). A final extension step was performed for 10min, at 72°C. The PCR product was digested with EcoRI and XhoI and gel purified. The PCR product was ligated into EcoRI and XhoI sites of pGEX-4T-1 expression vector (GE Healthcare Lifesciences, Uppsala, Sweden), which was a different vector as previously described in Suntravat et al (2013) (link). The ligated plasmid was transformed into E. coli Top10 competent cells (Invitrogen, CA, USA). Plasmid was extracted using the GenElute plasmid miniprep kit (Sigma-Aldrich, MO, USA). Plasmids containing inserts of the predicted size for Cam-dis were performed by PCR and further confirmed by sequencing for construction of in-frame.
Suntravat M., Barret H.S., Jurica C.A., Lucena S.E., Perez J.C, & Sánchez E.E. (2015). Recombinant disintegrin (r-Cam-dis) from Crotalus adamanteus inhibits adhesion of human pancreatic cancer cell lines to laminin-1 and vitronectin. Journal of Venom Research, 6, 1-10.
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3

Baculoviral Expression of Murine ITK and Rat PLCγ1

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Baculoviral expression constructs of full-length ITK (mouse) and rat full-length PLCγ1 (rat) have been described previously (44 (link)). The fragments of PLCγ1 SH2C, SH2C-linker and P-SH2N-SH2C-linker-SH3-H were amplified by polymerase chain reaction and cloned to pGEX-4T-1 expression vector (GE Healthcare). The SH2C R694A mutation was introduced by site directed mutagenesis (Stratagene). All sequences were verified by sequencing at the Iowa State University DNA synthesis and sequencing facility.
Devkota S., Joseph R.E., Min L., Fulton D.B, & Andreotti A. (2015). Scaffold protein SLP-76 primes PLCγ1 for activation by ITK mediated phosphorylation. Journal of molecular biology, 427(17), 2734-2747.
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4

Purification of Rab GEF Proteins

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GST-conjugates of GLUP6/GEF, Rab5-GEF2, GLUP4/Rab5a, Rab5b, Rab5c, Rab11, and truncated proteins of GEFs were produced in Escherichia coli BL21 (DE3) using the pGEX 4T-1 expression vector (GE Healthcare) and purified according to the procedure described earlier (Goh et al., 2007 (link)). The primer sequences for construction of the expression plasmids are shown in Supplementary Table S1 and the experimental procedures are described in the table legend. All Rab proteins were purified in the GDP-bound form in the presence of Mg2+ and without EDTA or GDP. After purification of GST-tagged proteins by chromatography on a Glutathione-Sepharose 4B column (GE Healthcare), the purified proteins were loaded onto a desalting column (GE healthcare) to remove glutathione and then stored frozen at −80ºC. The purification procedure was completed in one day to maximize enzyme activity.
Wen L., Fukuda M., Sunada M., Ishino S., Ishino Y., Okita T.W., Ogawa M., Ueda T, & Kumamaru T. (2015). Guanine nucleotide exchange factor 2 for Rab5 proteins coordinated with GLUP6/GEF regulates the intracellular transport of the proglutelin from the Golgi apparatus to the protein storage vacuole in rice endosperm. Journal of Experimental Botany, 66(20), 6137-6147.
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5

Recombinant TbLysoPLA Antibody Production

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A recombinant T. brucei full length LysoPLA fused to a GST tag on its N-terminal extremity, was expressed in E. coli One Shot BL21star (DE3) (Thermofisher) using the pGEX4T1 expression vector (GE Healthcare). Protein expression was induced at 37 °C for 3 h using 0.5 mM isopropyl-d-thiogalactopyranoside. The cells were harvested by centrifugation, resuspended in PBS and sonicated. Proteins released in the soluble form were purified using Glutathione Sepharose 4B according to the manufacturer’s instructions (GE Healthcare). On-column thrombin digestion was performed to release the protein without the GST tag, then dialysed against PBS. Purified recombinant TbLysoPLA was used as an antigen to raise polyclonal antibodies. Two rabbits were injected 4 times at 15-days intervals using Covalab facilities (www.covalab.com).
Monic S.G., Lamy A., Thonnus M., Bizarra-Rebelo T., Bringaud F., Smith T.K., Figueiredo L.M, & Rivière L. (2022). A novel lipase with dual localisation in Trypanosoma brucei. Scientific Reports, 12, 4766.
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6

Overexpression of Irc3 in E. coli

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Irc3 was overproduced in E. coli using pGEX4T-1 expression vector (GE Healthcare) as described previously23 (link).
Gaidutšik I., Sedman T., Sillamaa S, & Sedman J. (2016). Irc3 is a mitochondrial DNA branch migration enzyme. Scientific Reports, 6, 26414.
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7

Purification of GST-tagged UbcH10 protein

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The PCR product was cloned into pGEX-4T1 expression vector (GE Healthcare), leading to a protein with a cleavable N-terminal GST tag (GST-UbcH10). E. coli BL21 (DE3) RP strain was transformed with the recombinant plasmid for GST-UbcH10. Overnight cultures were used to inoculate 500 ml LB medium containing 50 µg/ml ampicillin, and protein induction was performed by the addition of 1 mM IPTG at 22°C when an OD600 value of 0.7 was reached. After approximately 16 h the cells were harvested and the proteins were isolated by sonicating cell pellets resuspended in 30 ml PBS1X in the presence of an EDTA free protease inhibitor cocktail (Roche Diagnostics). The crude cell extract was cleared by centrifugation at 18000 rpm and the supernatant was loaded onto a 1 ml GST-trap column connected to AKTA FPLC system (GE-Healthcare) equilibrated with binding buffer PBS1X. After washing with ten volumes of binding buffer, a single elution step was performed with 50 mM TrisHCl, 10 mM reduced glutathione. The fractions containing GST-UbcH10 were pooled and extensively dialyzed against PBS1X at 4°C. The homogeneity of the protein was tested by SDS–PAGE and mass spectrometry.
Correale S., de Paola I., Morgillo C.M., Federico A., Zaccaro L., Pallante P., Galeone A., Fusco A., Pedone E., Luque F.J, & Catalanotti B. (2014). Structural Model of the hUbA1-UbcH10 Quaternary Complex: In Silico and Experimental Analysis of the Protein-Protein Interactions between E1, E2 and Ubiquitin. PLoS ONE, 9(11), e112082.
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8

Recombinant Metal-Metallothionein Complexes

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Production and purification of recombinant metal-MT complexes of NpoMT1, FcaMT1, αNpoMT1 and δFcaMT1 domains were performed as described elsewhere [37 (link),50 (link)]. In brief, synthetic cDNAs codifying the different constructs were provided by Synbio Technologies (Monmouth Junction, NJ, USA), cloned in the pGEX-4T-1 expression vector (GE Healthcare, Chicago, IL, USA) and transformed in protease-deficient E. coli BL21 strain. Metal-MT complexes were produced in E. coli BL21 cultures expressing the recombinant plasmids, after induction with isopropyl-β-D-thiogalactopyranoside (100 μM) and supplementation with ZnCl2 (300 μM), CdCl2 (300 μM) or CuSO4 (500 μM). Metal-MT complexes were purified from the soluble protein fraction of sonicated bacteria by affinity purification of the GST-tagged proteins, and digestion with thrombin. The metal-MT complexes were concentrated with a 3 kDa Centripep Low Concentrator (Amicon, Merck), and fractionated on a Superdex-75 FPLC column (GE Healthcare) equilibrated with 20 mM Tris-HCl, pH 7.0 and run at 0.8 mL min−1. The protein-containing fractions, identified by their absorbance at 254 nm, were pooled and stored at −80 °C until use.
Calatayud S., Garcia-Risco M., Pedrini-Martha V., Niederwanger M., Dallinger R., Palacios Ò., Capdevila M, & Albalat R. (2022). The Modular Architecture of Metallothioneins Facilitates Domain Rearrangements and Contributes to Their Evolvability in Metal-Accumulating Mollusks. International Journal of Molecular Sciences, 23(24), 15824.
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9

Purification of Jmjd2a Jumonji Domain

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Jumonji domain containing region of Jmjd2a gene was cloned, expressed, and purified as described earlier [16] (link), [17] (link), with minor modifications. In brief, the N-terminal GST tag containing fusion Jmjd2a enzyme in pGEX-4T1 expression vector (GE Healthcare, Piscataway, NJ) was purified from Escherichia coli BL21 (DE3) cells, using affinity chromatography. The chromatographic fractions containing purified Jmjd2a enzyme was dialyzed in 25 mM NaCl (Sigma–Aldrich, St. Louis, MO), 25 mM HEPES (Sigma–Aldrich), pH 7.5 for ≈8 h. The dialyzed Jmjd2a protein was stored in 15% glycerol at –80 °C.
Vavilala D.T., Reddy S., S.a.c.h.c.h.i.d.a.n.a.n.d., Prakash S., Ponnaluri V.K., Kumar A, & Mukherji M. (2014). Prohexadione, a plant growth regulator, inhibits histone lysine demethylases and modulates epigenetics. Toxicology Reports, 1, 1152-1161.
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10

Cloning and Expression of E. coli LsrK and HPr Proteins

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The E. coli lsrK gene contained the sequence of the Bam HI restriction enzyme site, and thus, the lsrK gene was amplified from the genomic DNA of the E. coli K strain using the 5′- and 3′-primers with the Bgl II and Xho I restriction enzyme sites, respectively. The polymerase chain reaction (PCR) product was cloned into the pGEX-4T-1 expression vector (GE Healthcare) using the Bam HI and Xho I sites (21 (link)). For the coexpression of the N-terminal His-tagged LsrK and the native HPr proteins simultaneously, the lsrK and ptsH genes were cloned into the pACYCDuet-1 expression vector (Novagen). The lsrK gene was inserted into the first multiple cloning site (MCS) of the vector by using the Bam HI and Not I sites, and the ptsH gene was inserted into the second MCS by using the Nde I and Xho I sites, respectively.
To express the E. coli EI and HPr proteins as an intact form, the ptsI and ptsH genes were cloned into the pET-11a vector (37 (link)). The ptsH gene was additionally cloned into the pET-24a vector using the Nde I and Xho I sites to express the C-terminal six-His–tagged (C-His) HPr protein. The mutations resulting in the mutants H15E and H15A were introduced using the QuikChange Site-Directed Mutagenesis Kit (Stratagene).
Ha J.H., Hauk P., Cho K., Eo Y., Ma X., Stephens K., Cha S., Jeong M., Suh J.Y., Sintim H.O., Bentley W.E, & Ryu K.S. (2018). Evidence of link between quorum sensing and sugar metabolism in Escherichia coli revealed via cocrystal structures of LsrK and HPr. Science Advances, 4(6), eaar7063.
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