Complementary DNAs (cDNAs) of the human genes eEF1A, eEF1Bα, eEF1Bγ, eRF1, and eRF3 were obtained by reverse transcription followed by PCR (RT-PCR) using human placenta RNA (Clontech). DNA primers for RT-PCR were chosen based on the reported sequences (GenBankTM accession numbers NM_001402 for eEF1A, X60489 for eEF1Bα, Z11531 for eEF1Bγ, NM_004730 for eRF1, and NM_002094 for eRF3. eEF1A and eEF1Bγ cDNAs were cloned into the EMCV IRES-dependent expression vector pUC-T7-EMCV-His-MCS-ter (20 (link)) to generate pUC-T7-EMCV-His-eEF1A and pUC-T7-EMCV-His-eEF1Bγ, respectively. The eEF1Bα cDNA was cloned into pUC-T7-EMCV-MCS-ter (20 (link)) to construct pUC-T7-EMCV-eEF1Bα. eRF1 and eRF3 cDNAs were cloned into pGEX6P (GE Healthcare) to construct pGEX6P-eRF1 and pGEX6P-eRF3, respectively.
Pgex 6p
Manufactured by GE Healthcare
Sourced in United States
The PGEX-6P is a lab equipment product designed for protein expression and purification. It functions as a vector that enables the cloning and expression of recombinant proteins in Escherichia coli (E. coli) cells.
Automatically generated - may contain errors
Lab products found in correlation
Prescission protease, by GE Healthcare (2 mentions)
Pgex 6p 1, by GE Healthcare (2 mentions)
Reflex 3, by Bruker (1 mentions)
Phusion dna polymerase, by Thermo Fisher Scientific (1 mentions)
E coli bl21, by GE Healthcare (1 mentions)
Glutathion sepharose 4b, by GE Healthcare (1 mentions)
Quikchange technique, by Agilent Technologies (1 mentions)
Glutathione sepharose bead, by GE Healthcare (1 mentions)
Colorimetric assay, by Bio-Rad (1 mentions)
Chymotrypsin, by Merck Group (1 mentions)
Pet16b, by Merck Group (1 mentions)
Pmcherry, by Takara Bio (1 mentions)
Pgex 6p 2, by GE Healthcare (1 mentions)
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Pqcxip, by Takara Bio (1 mentions)
11 protocols using pgex 6p
1
Cloning of Human Eukaryotic Translation Factors
2
Recombinant expression and characterization of viral terminase proteins
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Synthetic genes encoding PaP3 TerS (Gene ID: 2700603, or orf1), NV1 TerS (Gene ID: 40099729) and LUZ24 TerS (Gene ID: 5896731) were purchased from Genewiz and ligated between BamHI and XhoI restriction sites of the expression vector pGEX-6P (GE Healthcare) (plasmid pGEX-6P-TerS). PaP3 TerL (Gene ID: 2700601, or orf3), also synthesized by Genewiz, was ligated in a pET28a (Novagen) expression vector between BamHI and XhoI (plasmid pET28a_PaP3-TerL). PaP3 ΔC122-TerS was constructed by introducing a stop codon at position E122 of TerS (plasmid pGEX-6P-ΔCTerS). Ala mutants DM-TerS (K17/K19), TM-TerS (K17/K19/K33) and pAla-TerS (K17/K19/K33/R49/R56/K57) were generated using site-directed mutagenesis. All plasmids were sequenced to confirm the fidelity of the DNA sequence. Eurofins Genomics LLC synthesized DNA fragments corresponding to the PaP3 cohesive (cos) site (5′-GCCGGCCCCTTTCCGCGTTA-3′) and complementary fragment, both 5′ Cy3-labeled. The single-stranded complementary cos oligos were annealed to generate double-stranded DNA (dsDNA). A non-specific 5′ Cy3-labeled 24-bp dsDNA (5′-GCACTGCAGTAACTTGTCAGTCAT-3′) generated from single-stranded oligos was used as a negative control.
3
Cloning and Purification of TNRC6A cNLS and Importin-α Constructs
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GST-TNRC6A cNLS (residues 1164–1172 of mouse TNRC6A corresponding to residues 1179–1187 of the human protein; numbering according to hTNRC6A isoform 1) and mutants were cloned into pGEX-6P (GE Healthcare) using overlapping oligonucleotides. GST-cNLS fusions were expressed in BL21(DE3) (NEB) cells overnight at 20°C following isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. Mouse Impα1 lacking the IBB domain (mImpα1ΔIBB; residues 70–529; also known as KPNA2) was expressed and purified as previously reported [43 ]. mImpα1ΔIBB mutants were obtained by site-directed mutagenesis and expressed and purified using similar methods as the wild-type protein. Human Impα1ΔIBB (hImpα1ΔIBB) was expressed in BL21(DE3) (NEB) cells in LB media at 25°C for 5 hours after induction with IPTG, and purified using similar techniques. The TNRC6 cNLS: mImpα1ΔIBB complex was isolated as described previously [44 (link)].
4
Affinity Purification and Mass Spectrometry of GST-Nax
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GST-Nax is a GST fusion protein at the C-terminus (amino acid residues 1489–1681) of mouse Nax (GenBank accession no. NM_009135). pGEX-Nax was prepared by subcloning Nax cDNA from pTRE-mNax [14 (link)] into pGEX-6P (GE Healthcare) to express GST-Nax. The GST-Nax protein was expressed in the E. coli strain BL21, and purified by glutathione affinity chromatography as described previously [15 (link)].
In pull-down experiments, glutathione Sepharose beads (20 μl) were coated with GST fusion proteins (2 μg), and then incubated overnight at 4°C with synaptosomal lysate (200 μg protein) prepared from the adult rat cerebrum, as described previously [17 (link)]. After washing the beads, the bound proteins were solubilized, separated by SDS-PAGE, and stained with Coomassie Brilliant Blue. Specific bands were excised, subjected to in-gel tryptic digestion, and then applied to matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (Reflex III, Bruker Daltonics). Peptide mass fingerprinting was performed by a Mascot search (http://www.matrixscience.com/ ) against the NCBI nonredundant protein database.
In pull-down experiments, glutathione Sepharose beads (20 μl) were coated with GST fusion proteins (2 μg), and then incubated overnight at 4°C with synaptosomal lysate (200 μg protein) prepared from the adult rat cerebrum, as described previously [17 (link)]. After washing the beads, the bound proteins were solubilized, separated by SDS-PAGE, and stained with Coomassie Brilliant Blue. Specific bands were excised, subjected to in-gel tryptic digestion, and then applied to matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (Reflex III, Bruker Daltonics). Peptide mass fingerprinting was performed by a Mascot search (
5
Cloning of Protein Expression Constructs
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All plasmids used in this study are listed and described in S1 Table . For cloning, open reading frames were amplified with Phusion DNA polymerase (Thermo Scientific) using primers harbouring relevant restriction sites. Amplicons were thereafter cloned into either the bacterial expression vectors pET28a (Novagen) and pGEX-6p (GE Healthcare) or the yeast expression vector pYES260 (Euroscarf) using indicated restriction enzymes (NEB) and T4 DNA ligase (NEB). Site directed mutagenesis was performed using the QuickChange method (Stratagene). All constructs were verified by DNA sequencing.
6
Recombinant Expression of Importins and Influenza Proteins
7
Purification and Pulldown of GST-Fusion Proteins
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For purification of GST-fusion proteins, the GST expression vector (pGex-6P, GE Healthcare) as well as the Flag-expression vector pCMV-Tag2B, both containing the respective insert (murine PID1; wild type and mutated LRP1 ICD; see Figure S2 for structure and mutations of the intracellular LRP1 domain) were transformed into E. coli BL21 (Stratagene). GST fusion proteins were purified via Glutathion Sepharose 4B (GE Healthcare). LRP1-ICDs were released from the column by incubation with PreScission protease (GE Healthcare). Integrity of all recombinant proteins was confirmed by SDS-PAGE. For pulldown analysis either cell or tissue lysates (150 μg) were incubated over night at 4 °C with GST-fusion proteins. Subsequently pulldown and unbound fractions were separated by centrifugation for 3 min at 9000 rpm. Supernatants were harvested (unbound fraction) and sepharose pellets were washed six times using PBS. Finally, pellets were reduced, and PID1-interacting proteins were analysed by Western blotting.
8
Recombinant Enzyme Purification and Mutagenesis
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Recombinant enzyme was prepared essentially as described previously [12 (link)]. Protein from PIP4K2C (UniGene 6280511) or associated mutants, cloned into the expression vector pGEX6P (GE Healthcare) was expressed and purified from Escherichia coli BL21(DE3). Cultures were induced with 0.4 mM IPTG and probe-sonicated in the presence of protease inhibitors. GST fusion proteins of PI5P4Kγ and PI5P4Kγ+, a mutant with specific activity close to that of the active PI5P4Kα isoform [12 (link)], were harvested by binding to glutathione sepharose beads (GE Healthcare) and cleaved in situ with 50 units of PreScission protease (GE Healthcare) for 4 h at 4°C. Purity was confirmed by SDS/PAGE and protein concentration determined by colorimetric assay (Bio-Rad). Site-directed mutagenesis using the QuikChange technique (Agilent Technologies) was used to generate clones from which mutant forms of PI5P4Kγ and PI5P4Kγ+ were produced (for mutagenesis primers see Supplementary Table S1).
9
Cloning and Proteolysis of Gp16 Protein
10
Construction and Characterization of Fluorescent Fusion Proteins
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pET-101-talin was constructed by PCR amplification of the human complementary DNA (cDNA) and direct subcloning of the resulting cDNA in the pET101/D-TOPO plasmid (Invitrogen). To generate the plasmid pET-101-talin-EGFP encoding for talin fused with EGFP at its C-terminus, the cDNA of EGFP was inserted into the AgeI site of pET-101-talin.
pGEX-6P2-mCherry-α-actinin-1, pGEX-6P2-α-actinin-1, pGEX-6P1-EGFP-vinculin-1-851 (Vh) and pGEX-6P1-EGFP-full-length-vinculin were constructed by PCR amplification of the human cDNAs and subcloning of the resulting cDNAs in the EcoRI and NotI sites of pGEX-6P2-mCherry, EcoRI and NotI sites of pGEX-6P2 (GE Healthcare), and SalI and NotI sites of pGEX-6P1-EGFP (for vinculin 1–851 and full-length).
pGEX-6P1-EGFP was a gift of Jan Faix (Hanover). pGEX-6P2-mCherry was constructed by PCR amplification of pmCherry (Clontech) and subcloning of the mCherry cDNA in pGEX-6P2 (GE Healthcare). All the constructs have been verified by sequencing.
pGEX-6P2-mCherry-α-actinin-1, pGEX-6P2-α-actinin-1, pGEX-6P1-EGFP-vinculin-1-851 (Vh) and pGEX-6P1-EGFP-full-length-vinculin were constructed by PCR amplification of the human cDNAs and subcloning of the resulting cDNAs in the EcoRI and NotI sites of pGEX-6P2-mCherry, EcoRI and NotI sites of pGEX-6P2 (GE Healthcare), and SalI and NotI sites of pGEX-6P1-EGFP (for vinculin 1–851 and full-length).
pGEX-6P1-EGFP was a gift of Jan Faix (Hanover). pGEX-6P2-mCherry was constructed by PCR amplification of pmCherry (Clontech) and subcloning of the mCherry cDNA in pGEX-6P2 (GE Healthcare). All the constructs have been verified by sequencing.
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