Structured Review

GE Healthcare pgex 4t 1
Bcr-Abl and C3G interact with Cbl, Abi1 and p130Cas. (A) Whole cell extracts from K562 were subjected to pull-down assays using GST-Abl-SH3 construct. The presence of C3G, Abi1, Cbl and p130Cas in the complexes was detected by inmunoblotting with specific antibodies. Representative Western blots of pull-down assays in K562 lysates using (B) Abi1-SH3 and SH3-b domains, (C) p130Cas-SH3, P1 and P2 domains, and (D) Cbl-SH3-b domain fused to GST as baits. The immunoblots were developed with specific antibodies against the indicated proteins. Glutathione-sepharose beads with whole cell lysate (B + K562), lysis buffer with the corresponding GST-fused proteins (LB) and <t>pGEX-4T-1</t> empty plasmid with K562 whole cell lysate (GST) were used as negative controls. L: whole cell lysate (40 μg). The asterisks indicate unspecific bands.
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Images

1) Product Images from "C3G forms complexes with Bcr-Abl and p38? MAPK at the focal adhesions in chronic myeloid leukemia cells: implication in the regulation of leukemic cell adhesion"

Article Title: C3G forms complexes with Bcr-Abl and p38? MAPK at the focal adhesions in chronic myeloid leukemia cells: implication in the regulation of leukemic cell adhesion

Journal: Cell Communication and Signaling : CCS

doi: 10.1186/1478-811X-11-9

Bcr-Abl and C3G interact with Cbl, Abi1 and p130Cas. (A) Whole cell extracts from K562 were subjected to pull-down assays using GST-Abl-SH3 construct. The presence of C3G, Abi1, Cbl and p130Cas in the complexes was detected by inmunoblotting with specific antibodies. Representative Western blots of pull-down assays in K562 lysates using (B) Abi1-SH3 and SH3-b domains, (C) p130Cas-SH3, P1 and P2 domains, and (D) Cbl-SH3-b domain fused to GST as baits. The immunoblots were developed with specific antibodies against the indicated proteins. Glutathione-sepharose beads with whole cell lysate (B + K562), lysis buffer with the corresponding GST-fused proteins (LB) and pGEX-4T-1 empty plasmid with K562 whole cell lysate (GST) were used as negative controls. L: whole cell lysate (40 μg). The asterisks indicate unspecific bands.
Figure Legend Snippet: Bcr-Abl and C3G interact with Cbl, Abi1 and p130Cas. (A) Whole cell extracts from K562 were subjected to pull-down assays using GST-Abl-SH3 construct. The presence of C3G, Abi1, Cbl and p130Cas in the complexes was detected by inmunoblotting with specific antibodies. Representative Western blots of pull-down assays in K562 lysates using (B) Abi1-SH3 and SH3-b domains, (C) p130Cas-SH3, P1 and P2 domains, and (D) Cbl-SH3-b domain fused to GST as baits. The immunoblots were developed with specific antibodies against the indicated proteins. Glutathione-sepharose beads with whole cell lysate (B + K562), lysis buffer with the corresponding GST-fused proteins (LB) and pGEX-4T-1 empty plasmid with K562 whole cell lysate (GST) were used as negative controls. L: whole cell lysate (40 μg). The asterisks indicate unspecific bands.

Techniques Used: Construct, Western Blot, Lysis, Plasmid Preparation

2) Product Images from "High yield production of pigeon circovirus capsid protein in the E. coli by evaluating the key parameters needed for protein expression"

Article Title: High yield production of pigeon circovirus capsid protein in the E. coli by evaluating the key parameters needed for protein expression

Journal: BMC Veterinary Research

doi: 10.1186/1746-6148-10-115

A schematic diagram of the constructions used in this study and the alignment results for the expressed PiCV capsid gene. (A) The full-length wild-type and codon-optimized PiCV capsid protein genes were cloned independently into three expression vectors pET28a, pGEX-4T-1 or pET32a. The PiCV capsid protein with the various different fusion tags, namely a six-histidine (6xHis), a Glutathione-S-transferase (GST) and a Thioredoxin-coupled six-histidine (Trx) at its N-terminus were expressed by T7 or Tac promoter-driving after IPTG induction. (B) The nucleotide sequences were compared between the wild-type (WT) and the codon-optimized (OPT) PiCV capsid protein genes. The asterisk (*) represents the fact that the aligned nucleotides are identical.
Figure Legend Snippet: A schematic diagram of the constructions used in this study and the alignment results for the expressed PiCV capsid gene. (A) The full-length wild-type and codon-optimized PiCV capsid protein genes were cloned independently into three expression vectors pET28a, pGEX-4T-1 or pET32a. The PiCV capsid protein with the various different fusion tags, namely a six-histidine (6xHis), a Glutathione-S-transferase (GST) and a Thioredoxin-coupled six-histidine (Trx) at its N-terminus were expressed by T7 or Tac promoter-driving after IPTG induction. (B) The nucleotide sequences were compared between the wild-type (WT) and the codon-optimized (OPT) PiCV capsid protein genes. The asterisk (*) represents the fact that the aligned nucleotides are identical.

Techniques Used: Clone Assay, Expressing

3) Product Images from "Recombinant Paracoccin Reproduces the Biological Properties of the Native Protein and Induces Protective Th1 Immunity against Paracoccidioides brasiliensis Infection"

Article Title: Recombinant Paracoccin Reproduces the Biological Properties of the Native Protein and Induces Protective Th1 Immunity against Paracoccidioides brasiliensis Infection

Journal: PLoS Neglected Tropical Diseases

doi: 10.1371/journal.pntd.0002788

Cloning strategies for cloning the paracoccin ORF for expression. The left panel shows the standard strategy, with PCR amplification of the largest exon, restriction endonuclease digestion, and cloning into the expression vector pGEX-4T-1. The positions of the exons are displayed on the map of the gene. Genomic DNA template was extracted from P. brasiliensis strain Pb18. Agarose gel electrophoresis (mid-left) shows the corresponding band amplified by PCR. The exon 4 amplicon was cloned by Bam HI and Eco RI digestion. The right panel shows the strategy for synthesis of the predicted paracoccin sequence fused with the 5′-UTR elements for transcription in the vector pUC57. Green arrow, T7 promoter; black box, lacO (lac operator); and red box, the phage T7 trailer sequence for ribosome binding.
Figure Legend Snippet: Cloning strategies for cloning the paracoccin ORF for expression. The left panel shows the standard strategy, with PCR amplification of the largest exon, restriction endonuclease digestion, and cloning into the expression vector pGEX-4T-1. The positions of the exons are displayed on the map of the gene. Genomic DNA template was extracted from P. brasiliensis strain Pb18. Agarose gel electrophoresis (mid-left) shows the corresponding band amplified by PCR. The exon 4 amplicon was cloned by Bam HI and Eco RI digestion. The right panel shows the strategy for synthesis of the predicted paracoccin sequence fused with the 5′-UTR elements for transcription in the vector pUC57. Green arrow, T7 promoter; black box, lacO (lac operator); and red box, the phage T7 trailer sequence for ribosome binding.

Techniques Used: Clone Assay, Expressing, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Agarose Gel Electrophoresis, Electrophoresis, Sequencing, Binding Assay

4) Product Images from "REGULATION OF PROGESTERONE RECEPTOR ACTIVITY BY CYCLIN DEPENDENT KINASES 1 AND 2 OCCURS IN PART BY PHOSPHORYLATION OF THE SRC-1 CARBOXYL-TERMINUS"

Article Title: REGULATION OF PROGESTERONE RECEPTOR ACTIVITY BY CYCLIN DEPENDENT KINASES 1 AND 2 OCCURS IN PART BY PHOSPHORYLATION OF THE SRC-1 CARBOXYL-TERMINUS

Journal:

doi: 10.1016/j.biocel.2011.04.009

Phosphorylation of GST-SRC-1 fragments enables identification of additional cyclin A2/Cdk2 target sites. (A) Schematic diagram depicting full length wild type SRC-1 (wt) and the eight fragments (F1–F8) generated by cloning into the pGEX-4T-1 vector.
Figure Legend Snippet: Phosphorylation of GST-SRC-1 fragments enables identification of additional cyclin A2/Cdk2 target sites. (A) Schematic diagram depicting full length wild type SRC-1 (wt) and the eight fragments (F1–F8) generated by cloning into the pGEX-4T-1 vector.

Techniques Used: Generated, Clone Assay, Plasmid Preparation

Related Articles

Clone Assay:

Article Title: Recombinant Paracoccin Reproduces the Biological Properties of the Native Protein and Induces Protective Th1 Immunity against Paracoccidioides brasiliensis Infection
Article Snippet: Therefore, paracoccin was cloned in two different ways. .. First, the largest exon was amplified from P. brasiliensis genomic DNA using the oligonucleotide primers FPADG ( 5′-CTGGATCCATGCAAGCACCCGACCAAC-3′ ) and RPADG ( 5′-CGGGAATTCCTACCAACTCGTTATTGATAGAGCGATAA-3′ ), and then cloned into pGEX-4T-1 (GE Healthcare, Upsala, Sweden) flanked by Bam HI and Eco RI restriction sites. .. Second, the full-length, predicted ORF encoding paracoccin was synthesized, and cloned into the pUC57 vector (GenScript, Piscataway, NJ, USA).

Article Title: High yield production of pigeon circovirus capsid protein in the E. coli by evaluating the key parameters needed for protein expression
Article Snippet: A 822 bp of cDNA fragment consisting of the cap gene that encodes the full-length PiCV capsid protein was synthesized by Genemark Biosci & Tech Co. (Taichung, Taiwan) based on the published sequence (Columbid circovirus, isolate 9030; Accession No. AJ298229). .. This cDNA was cloned into either pET28a, pET32a (Novagen, Madison, WI) or pGEX-4T-1 (GE Healthcare, Piscataway, NJ) individually using Eco R1 and Xho I (Takara, Japan) restriction sites. .. The resulting recombinant plasmids were designated pHis-Cap, pTrx-His-Cap and pGST-Cap, respectively (Figure , panel a, e and c).

Article Title: C3G forms complexes with Bcr-Abl and p38? MAPK at the focal adhesions in chronic myeloid leukemia cells: implication in the regulation of leukemic cell adhesion
Article Snippet: Constructs: Abi1-SH3, Abi1-SH3-b (SH3-binding), Cbl-SH3-b and p130Cas-SH3, p130Cas-P1 (proline-rich domain 1 or SH3-b1) and p130Cas-P2 (SH3-b2) domains were expressed as GST-fusion proteins. .. To do so, fragments were amplified by PCR and cloned into Eco RI-Xho I sites of pGEX-4T-1 (GE Healthcare Life Sciences). .. The oligos used were Abi1SH3-F: 5´-GGGGAATTCCCCAAGAATTATATTGAGAAAGTT-3´ and Abi1SH3-R: 5´-GGGCTCGAGTTAATCAGTATAGTGCATGATTGA-3; Abi1SH3b-F: 5´-GGGGAATTCCCCATTGCTGTGCCTACA-3´ and Abi1SH3b-R: 5´-GGGCTCGAGCAGCCTCCTCATCTTCAT-3´; CblSH3b-F: 5´-GAGGAATTCCCGCCTTCTCCATTCTCC-3´ and CblSH3b-R: 5´-GGGCTCGAGAGGTGGCAGTTTTGGCAC-3´; p130CasSH3-F: 5´-AGGGAATTCAACCACCTGAACGTGCTG-3´ and p130CasSH3-R: 5´-AGGCTCGAGGCCCACCAAGATCTTGAG-3; p130CasP1-F: 5´-AGGGAATTCGATAAGAAGCCAGCAGGG-3 and p130CasP1-R: 5´-AGGCTCGAGGTAGACGCTGTCTGGCTG-3´; p130CasP2-F: 5´-AGGGAATTCTCACTGCTCTTCAGACGG-3´ and p130CasP2-R: 5´-GGGCTCGAGGGTGAACTTAGGGGGTGA-3´.

Article Title: REGULATION OF PROGESTERONE RECEPTOR ACTIVITY BY CYCLIN DEPENDENT KINASES 1 AND 2 OCCURS IN PART BY PHOSPHORYLATION OF THE SRC-1 CARBOXYL-TERMINUS
Article Snippet: Eight fragments of SRC-1 (F1–F8) were amplified by PCR from the full length wild type (wt) pCR3.1-SRC-1 construct using Platinum Pfx polymerase (Invitrogen) and primers indicated in , with incorporation of BamHI and NotI restriction sites at the 5’ and 3’ ends, respectively. .. PCR products were ligated in frame into pGEX-4T-1 (GE Healthcare) and clones were verified by sequencing. .. Constructs were transformed into E. coli BL21 (DE3) cells (Stratagene, La Jolla, CA) and fusion protein expression was induced with 1 mM IPTG.

Article Title: F-Box Protein FBXO31 Mediates Cyclin D1 Degradation to Induce G1 Arrest Following DNA Damage
Article Snippet: The plasmid pLNCX2-FBXO31 was used as a template to generate FBXO31 mutants using the QuikChange XL site-directed mutagenesis kit (Stratagene). .. To generate in-frame GST-tagged fusion proteins, the wild type and mutant FBXO31 sequences were PCR amplified (using primers WT1_forward 5’-CGGGATCCGCTGCCACAGTGGAGTGC-3’ and WT1_reverse 5’-GGAATTCGATGAGGTCGTCGGGGCG-3’, or WT2_forward 5’-CGGAATTCCGG ATCCAGCTGCCCGAC-3’ and WT2_reverse 5’-ACGTCGACCACGGGCAGCACG AACGG-3’), digested with BamH1 and EcoR1, and cloned into pGEX-4T-1 (Amersham). .. In vitro ATM kinase assays were performed as described previously .

Article Title: Bioengineered bacterial vesicles as biological nano-heaters for optoacoustic imaging
Article Snippet: We anticipate that the OMVs presented in this work will be a starting point for future theranostic studies focused on simultaneous optoacoustic imaging and cancer therapeutics. .. Plasmid construction, bacterial strain and growth The melA gene of Rhizobium etli encoding tyrosinase (kindly provided by Professor Guillermo Gosset, Universidad Nacional Autónoma de México, Mexico) was cloned into pGEX-4T-1 (GE Healthcare, Freiburg, Germany). .. The construct pGEX-4T-1-melA was transformed into msbB mutant W3110-K12 E. coli (kindly provided by Professor Sangyong Jon, KAIST, South Korea) .

Article Title: Analysis of Metal-Binding Features of the Wild Type and Two Domain-Truncated Mutant Variants of Littorina littorea Metallothionein Reveals Its Cd-Specific Character
Article Snippet: Paragraph title: 3.2. Construction and Cloning of the cDNAs Encoding the LlwtMT, Lltr1MT, and Lltr2MT Proteins ... After PCR amplification (35 cycles: 95 °C 30 s, 50 °C 30 s, and 72 °C 30 s, using Expand High Fidelity (Roche Diagnostics S.L:, San Cugat del Vallès, Spain) thermostable DNA polymerase) of the synthetic cDNAs using the flanking primers 5′-TTTATTGGATCCATGAGCTC-3′ (LlwtMT forward); 5′-TTTCTCGAGTCATTTGCATG-3′ (LlwtMT reverse); 5′-TTTATTGGATCCGGTAAGGG-3′ (Lltr1MT forward); 5′-TTTCTCGAGTTACTTACAGG-3′ (Lltr1MT reverse); 5′-TTTATTGGATCCGGCAAAGGG-3′ (Lltr2MT forward); and 5′-TTTCTCGAGTTATTTGCAAG-3′ (Lltr2MT reverse), they were subsequently digested with BamHI and XhoI restriction enzymes, and the resulting products were ligated in-frame (DNA ligation kit, Takara Bio, Kusatsu, Shiga, Japan) in the pGEX-4T-1 (Amersham-GE Healthcare Europe, Cerdanyola del Valles, Spain) E. coli expression vector, which yields GST-fusion proteins.

Article Title: CIB1 is a Regulator of Pathological Cardiac Hypertrophy
Article Snippet: Cotransformation of pGBKT7-CnBshort and pGAD-CnA served as a positive control. .. To generate GST fusion proteins, CIB1 cDNA sequences were amplified by PCR and cloned into pGEX-4T-1 (Amersham Pharmacia Biotech). .. The fusion proteins were expressed in E. coli BL21 cells (Invitrogen) and precipitated with glutathione-sepharose beads (GE Healthcare).

Article Title: Does Variation of the Inter-Domain Linker Sequence Modulate the Metal Binding Behaviour of Helix pomatia Cd-Metallothionein?
Article Snippet: Paragraph title: 3.1. Construction and Cloning of the cDNAs Encoding the HpCdMT Linker Mutants ... They were subsequently digested with BamH I and Xho I restriction enzymes, and the resulting products were ligated in-frame (DNA ligation kit, Takara Bio, Kusatsu, Shiga, Japan) in the pGEX-4T-1 (Amersham-GE Healthcare Europe, Cerdanyola del Valles, Spain) E. coli expression vector, which yields GST-fusion proteins.

Article Title: Plasmodium falciparum ookinete expression of plasmepsin VII and plasmepsin X
Article Snippet: PCR products were gel purified on a 0.8 % agarose gel using the PureLink gel extraction kit (Invitrogen, Carlsbad, CA, USA), ligated into pCR4-TOPO (Invitrogen, Carlsbad, CA, USA), transformed into Top10 competent cells (Invitrogen, Carlsbad, CA, USA) and sequence-verified (Eton Bioscience, San Diego, CA, USA). .. PfPM VII and PfPM X genes were then cloned into expression vectors pET32 and pGEX 4T-1 (GE Healthcare, Piscataway, NJ, USA), respectively. .. The recombinant PfPM VII-HIS-tagged (rPfPM VII-HIS) and PfPM X-GST-tagged (rPfPM X-GST) fusion proteins were expressed in Rosetta (Merck/Novagen, Darmstadt, Germany) competent cells according to standard protocol [ ].

Article Title: Full length RTN3 regulates turnover of tubular endoplasmic reticulum via selective autophagy
Article Snippet: Resolution for MS/MS spectra was set to 30,000 at 200 m/z, AGC target to 1E5, max injection time to 64 ms and the isolation window to 1.6 Th. .. LC3s, GABARAPs and Ub proteins were cloned, as GST fusion proteins, into pGEX-4T-1 (GE Healthcare; Little Chalfont, UK) and expressed in Escherichia coli BL21 (DE3) cells in LB medium. .. Expression was induced by addition of 0.5 mM IPTG and cells were incubated at 37°C for 5 hr.

Article Title: Nuclear GSK3β promotes tumorigenesis by phosphorylating KDM1A and inducing its deubiquitination by USP22
Article Snippet: Site-directed mutagenesis in KDM1A was introduced using the QuikChange site-directed mutagenesis kit (Agilent Technologies). .. GST-tagged KDM1A or KDM1A S683A/S687A was constructed by cloning the wild-type or mutant cDNA into pGEX-4T-1 (GE Healthcare). .. Lentiviral expression of KDM1A-shR, KDM1A S683D, or KDM1A S683A was achieved by cloning the cDNA into a pLVX-IRES-Hyg vector.

Article Title: ESCRT-III mediates budding across the inner nuclear membrane and regulates its integrity
Article Snippet: ALIX cDNA was cloned into pcDNA3.1( + ) (Invitrogen, Carlsbad, CA) in frame with a Flag-epitope to generate pcDNA3.1-Flag-ALIX. pcDNA3.1-Flag-ALIX-I212D was constructed by replacing ALIX codons I212 in pcDNA3.1-Flag-ALIX with aspartic acid codons as reported previously . .. To generate fusion proteins of GST with the ALIX Bro domain (amino acids 1–358) and the ALIX V domain (amino acids 362–702) (GST-ALIX-Bro and GST-ALIX-V, respectively), pGEX-ALIX-Bro and pGEX-ALIX-V were constructed by cloning the appropriate ALI X domain, amplified by PCR from pCI-FLAG-ALIX, and cloned into pGEX-4T-1 (GE Healthcare Bio-Sciences, Piscataway, NJ). .. Sense and antisense oligonucleotides were designed for insertion into the Bbs I site in the pX330 bicistronic expression vector expressing Cas9 and synthetic single-guide RNA (Addgene, Cambridge, MA) as follows: 5′-CACCGGGCGGCCCGACCCCCCAGG-3′ and 5′-AAACCCTGGGGGGTCGGGCCGCCC-3′ for CHMP4B, and 5′-CACCGCAGGCCCAGTACTGCCGCG-3′ and 5′-AAACCGCGGCAGTACTGGGCCTGC-3′ for ALIX to produce pX330-CHMP4B and pX330-ALIX, respectively.

Article Title: Human babesiosis: Indication of a molecular mimicry between thrombospondin domains from a novel Babesia microti BmP53 protein and host platelets molecules
Article Snippet: Paragraph title: Selection and cloning of BmP53 gene truncates into pGEX-4T-1 vector ... The PCR products were ligated to the Bam HI and Xho I restriction sites of the glutathione S-transferase (GST) fusion expression vector, pGEX-4T-1 (GE Healthcare Life Sciences, UK).

Article Title: Transcriptional regulation of autophagy-lysosomal function in BRAF-driven melanoma progression and chemoresistance
Article Snippet: Flag-tagged BRAFV600E was cloned into pCDH-CMV-MCS-EF1-puro backbone (System Biosciences; SBI) to generate lentiviral transfer constructs. shRNA-resistant TFEB mutants were generated using site-directed mutagenesis (Q5 0552S, New England Biolab). .. For expression of GST fusion in Escherichia coli BL21 cells, the full-length TFEB, pro-TGFβ, and its point mutants were cloned into the pGEX-4T-1 (Pharmacia Amersham) vector or the pET32a (EMD Biosciences) vector. .. The plasmids of ZKSCAN3-V5 was provided by Dr. Pinghui Feng, University of Southern California, USA.

Article Title: Precision Targeting of BFL-1/A1 and an ATM Co-dependency in Human Cancer
Article Snippet: Protein purity and identity was confirmed by Coomassie staining and western blot analysis using a mouse monoclonal anti-His6 tag antibody (Abcam). .. Recombinant anti-apoptotic BCL-XL ΔC (aa 1–212) and MCL-1ΔNΔC (aa 172–329) were cloned into the pGEX-4T-1 (GE Healthcare, N-terminal GST tag) expression vector, expressed in BL21(DE3) Escherichia coli (Sigma Aldrich), and purified as described previously ( ). .. Protein expression was induced using 0.5 mM IPTG overnight at 37°C.

Article Title: Plasmodium berghei Circumvents Immune Responses Induced by Merozoite Surface Protein 1- and Apical Membrane Antigen 1-Based Vaccines
Article Snippet: The Pfmsp119 , Pbmsp119 , Pyama1-D3 and Pbama1-D3 genes were excised from pBACsurf-PfMSP119 , pBACsurf-PbMSP119 , pBACsurf-PyAMA1-D3, pBACsurf-PbAMA1-D3, respectively, by digestion with EcoR I and Sma I. .. Each of these DNA fragments was cloned into the EcoR I/Sma I sites of pGEX-4T-1 (GE Healthcare UK Limited, Buckinghamshire, UK). .. Recombinant PfMSP119 , PbMSP119 , PyAMA1-D3 and PbAMA1-D3 created as GST fusion proteins (termed GST-PfMSP119 , GST-PbMSP119 , GST-PyAMA1-D3 and GST-PbAMA1-D3 respectively), were expressed in Escherichia coli and purified using GST affinity columns (GE Healthcare UK Limited) as described previously .

Centrifugation:

Article Title: Polarized Exocytosis Induces Compensatory Endocytosis by Sec4p-Regulated Cortical Actin Polymerization
Article Snippet: For purification of GST-fusion proteins from bacterial extracts, and their subsequent use in actin polymerization assays, coding sequences for SLA1 , LAS17 , SEC4 , and YPT1 were sub-cloned into Bam HI and Sal I sites in pGEX-4T-1 (GE Healthcare Life Sciences, Pittsburg, PA). .. For protein expression, plasmids were transformed into the Escherichia coli strain BL21 (DE3) RIL.

Article Title: Structural basis for recruiting and shuttling of the spliceosomal deubiquitinase USP4 by SART3
Article Snippet: The glutathione S-transferase (GST)-tagged SART3 NLS, comprising residues 601–649, was overexpressed in E. coli strain Rossetta 2(DE3) at 18°C from pGEX-4T-1 (GE Healthcare), and hexa-histidine tagged mouse importin-α2 (SwissProt entry P52293) comprising residues 70–498 (ImpA) was overexpressed separately from the same strain at 18°C from pET-28a. .. After harvesting, the two sets of cells were mixed, suspended in 50 mM Tris-HCl pH 8.0, 150 mM NaCl and 0.1 mM PMSF, and disrupted by sonication on the ice.

Article Title: Precision Targeting of BFL-1/A1 and an ATM Co-dependency in Human Cancer
Article Snippet: Recombinant anti-apoptotic BCL-XL ΔC (aa 1–212) and MCL-1ΔNΔC (aa 172–329) were cloned into the pGEX-4T-1 (GE Healthcare, N-terminal GST tag) expression vector, expressed in BL21(DE3) Escherichia coli (Sigma Aldrich), and purified as described previously ( ). .. Protein expression was induced using 0.5 mM IPTG overnight at 37°C.

Amplification:

Article Title: Recombinant Paracoccin Reproduces the Biological Properties of the Native Protein and Induces Protective Th1 Immunity against Paracoccidioides brasiliensis Infection
Article Snippet: Therefore, paracoccin was cloned in two different ways. .. First, the largest exon was amplified from P. brasiliensis genomic DNA using the oligonucleotide primers FPADG ( 5′-CTGGATCCATGCAAGCACCCGACCAAC-3′ ) and RPADG ( 5′-CGGGAATTCCTACCAACTCGTTATTGATAGAGCGATAA-3′ ), and then cloned into pGEX-4T-1 (GE Healthcare, Upsala, Sweden) flanked by Bam HI and Eco RI restriction sites. .. Second, the full-length, predicted ORF encoding paracoccin was synthesized, and cloned into the pUC57 vector (GenScript, Piscataway, NJ, USA).

Article Title: C3G forms complexes with Bcr-Abl and p38? MAPK at the focal adhesions in chronic myeloid leukemia cells: implication in the regulation of leukemic cell adhesion
Article Snippet: Constructs: Abi1-SH3, Abi1-SH3-b (SH3-binding), Cbl-SH3-b and p130Cas-SH3, p130Cas-P1 (proline-rich domain 1 or SH3-b1) and p130Cas-P2 (SH3-b2) domains were expressed as GST-fusion proteins. .. To do so, fragments were amplified by PCR and cloned into Eco RI-Xho I sites of pGEX-4T-1 (GE Healthcare Life Sciences). .. The oligos used were Abi1SH3-F: 5´-GGGGAATTCCCCAAGAATTATATTGAGAAAGTT-3´ and Abi1SH3-R: 5´-GGGCTCGAGTTAATCAGTATAGTGCATGATTGA-3; Abi1SH3b-F: 5´-GGGGAATTCCCCATTGCTGTGCCTACA-3´ and Abi1SH3b-R: 5´-GGGCTCGAGCAGCCTCCTCATCTTCAT-3´; CblSH3b-F: 5´-GAGGAATTCCCGCCTTCTCCATTCTCC-3´ and CblSH3b-R: 5´-GGGCTCGAGAGGTGGCAGTTTTGGCAC-3´; p130CasSH3-F: 5´-AGGGAATTCAACCACCTGAACGTGCTG-3´ and p130CasSH3-R: 5´-AGGCTCGAGGCCCACCAAGATCTTGAG-3; p130CasP1-F: 5´-AGGGAATTCGATAAGAAGCCAGCAGGG-3 and p130CasP1-R: 5´-AGGCTCGAGGTAGACGCTGTCTGGCTG-3´; p130CasP2-F: 5´-AGGGAATTCTCACTGCTCTTCAGACGG-3´ and p130CasP2-R: 5´-GGGCTCGAGGGTGAACTTAGGGGGTGA-3´.

Article Title: REGULATION OF PROGESTERONE RECEPTOR ACTIVITY BY CYCLIN DEPENDENT KINASES 1 AND 2 OCCURS IN PART BY PHOSPHORYLATION OF THE SRC-1 CARBOXYL-TERMINUS
Article Snippet: Eight fragments of SRC-1 (F1–F8) were amplified by PCR from the full length wild type (wt) pCR3.1-SRC-1 construct using Platinum Pfx polymerase (Invitrogen) and primers indicated in , with incorporation of BamHI and NotI restriction sites at the 5’ and 3’ ends, respectively. .. PCR products were ligated in frame into pGEX-4T-1 (GE Healthcare) and clones were verified by sequencing.

Article Title: F-Box Protein FBXO31 Mediates Cyclin D1 Degradation to Induce G1 Arrest Following DNA Damage
Article Snippet: The plasmid pLNCX2-FBXO31 was used as a template to generate FBXO31 mutants using the QuikChange XL site-directed mutagenesis kit (Stratagene). .. To generate in-frame GST-tagged fusion proteins, the wild type and mutant FBXO31 sequences were PCR amplified (using primers WT1_forward 5’-CGGGATCCGCTGCCACAGTGGAGTGC-3’ and WT1_reverse 5’-GGAATTCGATGAGGTCGTCGGGGCG-3’, or WT2_forward 5’-CGGAATTCCGG ATCCAGCTGCCCGAC-3’ and WT2_reverse 5’-ACGTCGACCACGGGCAGCACG AACGG-3’), digested with BamH1 and EcoR1, and cloned into pGEX-4T-1 (Amersham). .. In vitro ATM kinase assays were performed as described previously .

Article Title: Analysis of Metal-Binding Features of the Wild Type and Two Domain-Truncated Mutant Variants of Littorina littorea Metallothionein Reveals Its Cd-Specific Character
Article Snippet: The LlwtMT, Lltr1MT, and Lltr2MT coding sequences designed this way were purchased as synthetic DNAs IDT & Conda Labs (Madrid, Spain). .. After PCR amplification (35 cycles: 95 °C 30 s, 50 °C 30 s, and 72 °C 30 s, using Expand High Fidelity (Roche Diagnostics S.L:, San Cugat del Vallès, Spain) thermostable DNA polymerase) of the synthetic cDNAs using the flanking primers 5′-TTTATTGGATCCATGAGCTC-3′ (LlwtMT forward); 5′-TTTCTCGAGTCATTTGCATG-3′ (LlwtMT reverse); 5′-TTTATTGGATCCGGTAAGGG-3′ (Lltr1MT forward); 5′-TTTCTCGAGTTACTTACAGG-3′ (Lltr1MT reverse); 5′-TTTATTGGATCCGGCAAAGGG-3′ (Lltr2MT forward); and 5′-TTTCTCGAGTTATTTGCAAG-3′ (Lltr2MT reverse), they were subsequently digested with BamHI and XhoI restriction enzymes, and the resulting products were ligated in-frame (DNA ligation kit, Takara Bio, Kusatsu, Shiga, Japan) in the pGEX-4T-1 (Amersham-GE Healthcare Europe, Cerdanyola del Valles, Spain) E. coli expression vector, which yields GST-fusion proteins. .. DNA sequencing allowed confirming of all the DNA constructs (ABIPRISM 310, Applied Biosystems, Foster City, CA, USA) using the BigDye Terminator.

Article Title: CIB1 is a Regulator of Pathological Cardiac Hypertrophy
Article Snippet: Cotransformation of pGBKT7-CnBshort and pGAD-CnA served as a positive control. .. To generate GST fusion proteins, CIB1 cDNA sequences were amplified by PCR and cloned into pGEX-4T-1 (Amersham Pharmacia Biotech). .. The fusion proteins were expressed in E. coli BL21 cells (Invitrogen) and precipitated with glutathione-sepharose beads (GE Healthcare).

Article Title: Does Variation of the Inter-Domain Linker Sequence Modulate the Metal Binding Behaviour of Helix pomatia Cd-Metallothionein?
Article Snippet: After PCR amplification (35 cycles: 95 °C 30 s, 50 °C 30 s, and 72 °C 30 s, using Expand High Fidelity (Roche) thermostable DNA polymerase) of the synthetic cDNAs using the flanking primers: 5’-TTTATTGGATCCGGTAAGGG-3’ (HpCdMcMT forward); 5’-TTTCTCGAGTTACTTACAGG-3’ (HpCdMcMT reverse); 5’-TTTATTGGATCCGGCAAAGGG-3’ (HpCdPlMT forward); and 5’-TTTCTCGAGTTATTTGCAAG-3’ (HpCdPlMT reverse). .. They were subsequently digested with BamH I and Xho I restriction enzymes, and the resulting products were ligated in-frame (DNA ligation kit, Takara Bio, Kusatsu, Shiga, Japan) in the pGEX-4T-1 (Amersham-GE Healthcare Europe, Cerdanyola del Valles, Spain) E. coli expression vector, which yields GST-fusion proteins.

Article Title: Plasmodium falciparum ookinete expression of plasmepsin VII and plasmepsin X
Article Snippet: PfPM VII and PfPM X genes were amplified from genomic NF54 DNA to generate a gene that lacked the signal peptide. .. PfPM VII and PfPM X genes were then cloned into expression vectors pET32 and pGEX 4T-1 (GE Healthcare, Piscataway, NJ, USA), respectively.

Article Title: Nuclear GSK3β promotes tumorigenesis by phosphorylating KDM1A and inducing its deubiquitination by USP22
Article Snippet: GST-tagged KDM1A or KDM1A S683A/S687A was constructed by cloning the wild-type or mutant cDNA into pGEX-4T-1 (GE Healthcare). .. Lentiviral expression of KDM1A-shR, KDM1A S683D, or KDM1A S683A was achieved by cloning the cDNA into a pLVX-IRES-Hyg vector.

Article Title: ESCRT-III mediates budding across the inner nuclear membrane and regulates its integrity
Article Snippet: ALIX cDNA was cloned into pcDNA3.1( + ) (Invitrogen, Carlsbad, CA) in frame with a Flag-epitope to generate pcDNA3.1-Flag-ALIX. pcDNA3.1-Flag-ALIX-I212D was constructed by replacing ALIX codons I212 in pcDNA3.1-Flag-ALIX with aspartic acid codons as reported previously . .. To generate fusion proteins of GST with the ALIX Bro domain (amino acids 1–358) and the ALIX V domain (amino acids 362–702) (GST-ALIX-Bro and GST-ALIX-V, respectively), pGEX-ALIX-Bro and pGEX-ALIX-V were constructed by cloning the appropriate ALI X domain, amplified by PCR from pCI-FLAG-ALIX, and cloned into pGEX-4T-1 (GE Healthcare Bio-Sciences, Piscataway, NJ). .. Sense and antisense oligonucleotides were designed for insertion into the Bbs I site in the pX330 bicistronic expression vector expressing Cas9 and synthetic single-guide RNA (Addgene, Cambridge, MA) as follows: 5′-CACCGGGCGGCCCGACCCCCCAGG-3′ and 5′-AAACCCTGGGGGGTCGGGCCGCCC-3′ for CHMP4B, and 5′-CACCGCAGGCCCAGTACTGCCGCG-3′ and 5′-AAACCGCGGCAGTACTGGGCCTGC-3′ for ALIX to produce pX330-CHMP4B and pX330-ALIX, respectively.

Article Title: Human babesiosis: Indication of a molecular mimicry between thrombospondin domains from a novel Babesia microti BmP53 protein and host platelets molecules
Article Snippet: For cloning, two pairs of oligonucleotide primers with Bam HI and Xho I restriction enzyme sites were designed and used for the amplification of both truncates, BmP53tr1-TSP1 (forward primer: 5′-CGGGATCCGAGAAGATAAAGGATGAA-3′ ; reverse primer: 5′-CGCTCGAGTTATTTATAGCTAGAGCTCAG -3′ ) and BmP53tr2 (forward primer: 5′-CGGGATCCAGGTTAATGAAGCTGCCA-3′ ; reverse primer: 5′-CGCTCGAGTTACGCAAATTGGGCAAATGA-3′ ). .. The PCR products were ligated to the Bam HI and Xho I restriction sites of the glutathione S-transferase (GST) fusion expression vector, pGEX-4T-1 (GE Healthcare Life Sciences, UK).

Filtration:

Article Title: Precision Targeting of BFL-1/A1 and an ATM Co-dependency in Human Cancer
Article Snippet: The BFL-1 containing fraction was then dialyzed overnight, concentrated, and loaded onto a Superdex S-75 (GE Healthcare) gel filtration system. .. Recombinant anti-apoptotic BCL-XL ΔC (aa 1–212) and MCL-1ΔNΔC (aa 172–329) were cloned into the pGEX-4T-1 (GE Healthcare, N-terminal GST tag) expression vector, expressed in BL21(DE3) Escherichia coli (Sigma Aldrich), and purified as described previously ( ).

DNA Ligation:

Article Title: Analysis of Metal-Binding Features of the Wild Type and Two Domain-Truncated Mutant Variants of Littorina littorea Metallothionein Reveals Its Cd-Specific Character
Article Snippet: The LlwtMT, Lltr1MT, and Lltr2MT coding sequences designed this way were purchased as synthetic DNAs IDT & Conda Labs (Madrid, Spain). .. After PCR amplification (35 cycles: 95 °C 30 s, 50 °C 30 s, and 72 °C 30 s, using Expand High Fidelity (Roche Diagnostics S.L:, San Cugat del Vallès, Spain) thermostable DNA polymerase) of the synthetic cDNAs using the flanking primers 5′-TTTATTGGATCCATGAGCTC-3′ (LlwtMT forward); 5′-TTTCTCGAGTCATTTGCATG-3′ (LlwtMT reverse); 5′-TTTATTGGATCCGGTAAGGG-3′ (Lltr1MT forward); 5′-TTTCTCGAGTTACTTACAGG-3′ (Lltr1MT reverse); 5′-TTTATTGGATCCGGCAAAGGG-3′ (Lltr2MT forward); and 5′-TTTCTCGAGTTATTTGCAAG-3′ (Lltr2MT reverse), they were subsequently digested with BamHI and XhoI restriction enzymes, and the resulting products were ligated in-frame (DNA ligation kit, Takara Bio, Kusatsu, Shiga, Japan) in the pGEX-4T-1 (Amersham-GE Healthcare Europe, Cerdanyola del Valles, Spain) E. coli expression vector, which yields GST-fusion proteins. .. DNA sequencing allowed confirming of all the DNA constructs (ABIPRISM 310, Applied Biosystems, Foster City, CA, USA) using the BigDye Terminator.

Article Title: Does Variation of the Inter-Domain Linker Sequence Modulate the Metal Binding Behaviour of Helix pomatia Cd-Metallothionein?
Article Snippet: After PCR amplification (35 cycles: 95 °C 30 s, 50 °C 30 s, and 72 °C 30 s, using Expand High Fidelity (Roche) thermostable DNA polymerase) of the synthetic cDNAs using the flanking primers: 5’-TTTATTGGATCCGGTAAGGG-3’ (HpCdMcMT forward); 5’-TTTCTCGAGTTACTTACAGG-3’ (HpCdMcMT reverse); 5’-TTTATTGGATCCGGCAAAGGG-3’ (HpCdPlMT forward); and 5’-TTTCTCGAGTTATTTGCAAG-3’ (HpCdPlMT reverse). .. They were subsequently digested with BamH I and Xho I restriction enzymes, and the resulting products were ligated in-frame (DNA ligation kit, Takara Bio, Kusatsu, Shiga, Japan) in the pGEX-4T-1 (Amersham-GE Healthcare Europe, Cerdanyola del Valles, Spain) E. coli expression vector, which yields GST-fusion proteins. .. DNA sequencing allow confirming all the DNA constructs (ABIPRISM 310, Applied Biosystems, Foster City, CA, USA), using BigDye Terminator.

Synthesized:

Article Title: Recombinant Paracoccin Reproduces the Biological Properties of the Native Protein and Induces Protective Th1 Immunity against Paracoccidioides brasiliensis Infection
Article Snippet: First, the largest exon was amplified from P. brasiliensis genomic DNA using the oligonucleotide primers FPADG ( 5′-CTGGATCCATGCAAGCACCCGACCAAC-3′ ) and RPADG ( 5′-CGGGAATTCCTACCAACTCGTTATTGATAGAGCGATAA-3′ ), and then cloned into pGEX-4T-1 (GE Healthcare, Upsala, Sweden) flanked by Bam HI and Eco RI restriction sites. .. Second, the full-length, predicted ORF encoding paracoccin was synthesized, and cloned into the pUC57 vector (GenScript, Piscataway, NJ, USA).

Article Title: High yield production of pigeon circovirus capsid protein in the E. coli by evaluating the key parameters needed for protein expression
Article Snippet: A 822 bp of cDNA fragment consisting of the cap gene that encodes the full-length PiCV capsid protein was synthesized by Genemark Biosci & Tech Co. (Taichung, Taiwan) based on the published sequence (Columbid circovirus, isolate 9030; Accession No. AJ298229). .. This cDNA was cloned into either pET28a, pET32a (Novagen, Madison, WI) or pGEX-4T-1 (GE Healthcare, Piscataway, NJ) individually using Eco R1 and Xho I (Takara, Japan) restriction sites.

Article Title: CIB1 is a Regulator of Pathological Cardiac Hypertrophy
Article Snippet: To generate GST fusion proteins, CIB1 cDNA sequences were amplified by PCR and cloned into pGEX-4T-1 (Amersham Pharmacia Biotech). .. To generate GST fusion proteins, CIB1 cDNA sequences were amplified by PCR and cloned into pGEX-4T-1 (Amersham Pharmacia Biotech).

Construct:

Article Title: High yield production of pigeon circovirus capsid protein in the E. coli by evaluating the key parameters needed for protein expression
Article Snippet: This cDNA was cloned into either pET28a, pET32a (Novagen, Madison, WI) or pGEX-4T-1 (GE Healthcare, Piscataway, NJ) individually using Eco R1 and Xho I (Takara, Japan) restriction sites. .. The resulting recombinant plasmids were designated pHis-Cap, pTrx-His-Cap and pGST-Cap, respectively (Figure , panel a, e and c).

Article Title: C3G forms complexes with Bcr-Abl and p38? MAPK at the focal adhesions in chronic myeloid leukemia cells: implication in the regulation of leukemic cell adhesion
Article Snippet: Constructs: Abi1-SH3, Abi1-SH3-b (SH3-binding), Cbl-SH3-b and p130Cas-SH3, p130Cas-P1 (proline-rich domain 1 or SH3-b1) and p130Cas-P2 (SH3-b2) domains were expressed as GST-fusion proteins. .. To do so, fragments were amplified by PCR and cloned into Eco RI-Xho I sites of pGEX-4T-1 (GE Healthcare Life Sciences).

Article Title: REGULATION OF PROGESTERONE RECEPTOR ACTIVITY BY CYCLIN DEPENDENT KINASES 1 AND 2 OCCURS IN PART BY PHOSPHORYLATION OF THE SRC-1 CARBOXYL-TERMINUS
Article Snippet: Eight fragments of SRC-1 (F1–F8) were amplified by PCR from the full length wild type (wt) pCR3.1-SRC-1 construct using Platinum Pfx polymerase (Invitrogen) and primers indicated in , with incorporation of BamHI and NotI restriction sites at the 5’ and 3’ ends, respectively. .. PCR products were ligated in frame into pGEX-4T-1 (GE Healthcare) and clones were verified by sequencing.

Article Title: Full length RTN3 regulates turnover of tubular endoplasmic reticulum via selective autophagy
Article Snippet: LC3s, GABARAPs and Ub proteins were cloned, as GST fusion proteins, into pGEX-4T-1 (GE Healthcare; Little Chalfont, UK) and expressed in Escherichia coli BL21 (DE3) cells in LB medium. .. LC3s, GABARAPs and Ub proteins were cloned, as GST fusion proteins, into pGEX-4T-1 (GE Healthcare; Little Chalfont, UK) and expressed in Escherichia coli BL21 (DE3) cells in LB medium.

Article Title: Nuclear GSK3β promotes tumorigenesis by phosphorylating KDM1A and inducing its deubiquitination by USP22
Article Snippet: Site-directed mutagenesis in KDM1A was introduced using the QuikChange site-directed mutagenesis kit (Agilent Technologies). .. GST-tagged KDM1A or KDM1A S683A/S687A was constructed by cloning the wild-type or mutant cDNA into pGEX-4T-1 (GE Healthcare). .. Lentiviral expression of KDM1A-shR, KDM1A S683D, or KDM1A S683A was achieved by cloning the cDNA into a pLVX-IRES-Hyg vector.

Article Title: ESCRT-III mediates budding across the inner nuclear membrane and regulates its integrity
Article Snippet: ALIX cDNA was cloned into pcDNA3.1( + ) (Invitrogen, Carlsbad, CA) in frame with a Flag-epitope to generate pcDNA3.1-Flag-ALIX. pcDNA3.1-Flag-ALIX-I212D was constructed by replacing ALIX codons I212 in pcDNA3.1-Flag-ALIX with aspartic acid codons as reported previously . .. To generate fusion proteins of GST with the ALIX Bro domain (amino acids 1–358) and the ALIX V domain (amino acids 362–702) (GST-ALIX-Bro and GST-ALIX-V, respectively), pGEX-ALIX-Bro and pGEX-ALIX-V were constructed by cloning the appropriate ALI X domain, amplified by PCR from pCI-FLAG-ALIX, and cloned into pGEX-4T-1 (GE Healthcare Bio-Sciences, Piscataway, NJ). .. Sense and antisense oligonucleotides were designed for insertion into the Bbs I site in the pX330 bicistronic expression vector expressing Cas9 and synthetic single-guide RNA (Addgene, Cambridge, MA) as follows: 5′-CACCGGGCGGCCCGACCCCCCAGG-3′ and 5′-AAACCCTGGGGGGTCGGGCCGCCC-3′ for CHMP4B, and 5′-CACCGCAGGCCCAGTACTGCCGCG-3′ and 5′-AAACCGCGGCAGTACTGGGCCTGC-3′ for ALIX to produce pX330-CHMP4B and pX330-ALIX, respectively.

Article Title: Transcriptional regulation of autophagy-lysosomal function in BRAF-driven melanoma progression and chemoresistance
Article Snippet: Flag-tagged BRAFV600E was cloned into pCDH-CMV-MCS-EF1-puro backbone (System Biosciences; SBI) to generate lentiviral transfer constructs. shRNA-resistant TFEB mutants were generated using site-directed mutagenesis (Q5 0552S, New England Biolab). .. For expression of GST fusion in Escherichia coli BL21 cells, the full-length TFEB, pro-TGFβ, and its point mutants were cloned into the pGEX-4T-1 (Pharmacia Amersham) vector or the pET32a (EMD Biosciences) vector.

Enzyme-linked Immunosorbent Assay:

Article Title: Plasmodium berghei Circumvents Immune Responses Induced by Merozoite Surface Protein 1- and Apical Membrane Antigen 1-Based Vaccines
Article Snippet: Each of these DNA fragments was cloned into the EcoR I/Sma I sites of pGEX-4T-1 (GE Healthcare UK Limited, Buckinghamshire, UK). .. Each of these DNA fragments was cloned into the EcoR I/Sma I sites of pGEX-4T-1 (GE Healthcare UK Limited, Buckinghamshire, UK).

Polymerization Assay:

Article Title: Polarized Exocytosis Induces Compensatory Endocytosis by Sec4p-Regulated Cortical Actin Polymerization
Article Snippet: Paragraph title: In Vitro Pyrene-Actin Polymerization Assay and Affinity Purification of Fusion Proteins ... For purification of GST-fusion proteins from bacterial extracts, and their subsequent use in actin polymerization assays, coding sequences for SLA1 , LAS17 , SEC4 , and YPT1 were sub-cloned into Bam HI and Sal I sites in pGEX-4T-1 (GE Healthcare Life Sciences, Pittsburg, PA).

Expressing:

Article Title: High yield production of pigeon circovirus capsid protein in the E. coli by evaluating the key parameters needed for protein expression
Article Snippet: This cDNA was cloned into either pET28a, pET32a (Novagen, Madison, WI) or pGEX-4T-1 (GE Healthcare, Piscataway, NJ) individually using Eco R1 and Xho I (Takara, Japan) restriction sites. .. The resulting recombinant plasmids were designated pHis-Cap, pTrx-His-Cap and pGST-Cap, respectively (Figure , panel a, e and c).

Article Title: Analysis of Metal-Binding Features of the Wild Type and Two Domain-Truncated Mutant Variants of Littorina littorea Metallothionein Reveals Its Cd-Specific Character
Article Snippet: The LlwtMT, Lltr1MT, and Lltr2MT coding sequences designed this way were purchased as synthetic DNAs IDT & Conda Labs (Madrid, Spain). .. After PCR amplification (35 cycles: 95 °C 30 s, 50 °C 30 s, and 72 °C 30 s, using Expand High Fidelity (Roche Diagnostics S.L:, San Cugat del Vallès, Spain) thermostable DNA polymerase) of the synthetic cDNAs using the flanking primers 5′-TTTATTGGATCCATGAGCTC-3′ (LlwtMT forward); 5′-TTTCTCGAGTCATTTGCATG-3′ (LlwtMT reverse); 5′-TTTATTGGATCCGGTAAGGG-3′ (Lltr1MT forward); 5′-TTTCTCGAGTTACTTACAGG-3′ (Lltr1MT reverse); 5′-TTTATTGGATCCGGCAAAGGG-3′ (Lltr2MT forward); and 5′-TTTCTCGAGTTATTTGCAAG-3′ (Lltr2MT reverse), they were subsequently digested with BamHI and XhoI restriction enzymes, and the resulting products were ligated in-frame (DNA ligation kit, Takara Bio, Kusatsu, Shiga, Japan) in the pGEX-4T-1 (Amersham-GE Healthcare Europe, Cerdanyola del Valles, Spain) E. coli expression vector, which yields GST-fusion proteins. .. DNA sequencing allowed confirming of all the DNA constructs (ABIPRISM 310, Applied Biosystems, Foster City, CA, USA) using the BigDye Terminator.

Article Title: Does Variation of the Inter-Domain Linker Sequence Modulate the Metal Binding Behaviour of Helix pomatia Cd-Metallothionein?
Article Snippet: After PCR amplification (35 cycles: 95 °C 30 s, 50 °C 30 s, and 72 °C 30 s, using Expand High Fidelity (Roche) thermostable DNA polymerase) of the synthetic cDNAs using the flanking primers: 5’-TTTATTGGATCCGGTAAGGG-3’ (HpCdMcMT forward); 5’-TTTCTCGAGTTACTTACAGG-3’ (HpCdMcMT reverse); 5’-TTTATTGGATCCGGCAAAGGG-3’ (HpCdPlMT forward); and 5’-TTTCTCGAGTTATTTGCAAG-3’ (HpCdPlMT reverse). .. They were subsequently digested with BamH I and Xho I restriction enzymes, and the resulting products were ligated in-frame (DNA ligation kit, Takara Bio, Kusatsu, Shiga, Japan) in the pGEX-4T-1 (Amersham-GE Healthcare Europe, Cerdanyola del Valles, Spain) E. coli expression vector, which yields GST-fusion proteins. .. DNA sequencing allow confirming all the DNA constructs (ABIPRISM 310, Applied Biosystems, Foster City, CA, USA), using BigDye Terminator.

Article Title: Plasmodium falciparum ookinete expression of plasmepsin VII and plasmepsin X
Article Snippet: PCR products were gel purified on a 0.8 % agarose gel using the PureLink gel extraction kit (Invitrogen, Carlsbad, CA, USA), ligated into pCR4-TOPO (Invitrogen, Carlsbad, CA, USA), transformed into Top10 competent cells (Invitrogen, Carlsbad, CA, USA) and sequence-verified (Eton Bioscience, San Diego, CA, USA). .. PfPM VII and PfPM X genes were then cloned into expression vectors pET32 and pGEX 4T-1 (GE Healthcare, Piscataway, NJ, USA), respectively. .. The recombinant PfPM VII-HIS-tagged (rPfPM VII-HIS) and PfPM X-GST-tagged (rPfPM X-GST) fusion proteins were expressed in Rosetta (Merck/Novagen, Darmstadt, Germany) competent cells according to standard protocol [ ].

Article Title: Polarized Exocytosis Induces Compensatory Endocytosis by Sec4p-Regulated Cortical Actin Polymerization
Article Snippet: For purification of GST-fusion proteins from bacterial extracts, and their subsequent use in actin polymerization assays, coding sequences for SLA1 , LAS17 , SEC4 , and YPT1 were sub-cloned into Bam HI and Sal I sites in pGEX-4T-1 (GE Healthcare Life Sciences, Pittsburg, PA). .. For protein expression, plasmids were transformed into the Escherichia coli strain BL21 (DE3) RIL.

Article Title: Structural basis for recruiting and shuttling of the spliceosomal deubiquitinase USP4 by SART3
Article Snippet: Paragraph title: Protein expression and purification ... The glutathione S-transferase (GST)-tagged SART3 NLS, comprising residues 601–649, was overexpressed in E. coli strain Rossetta 2(DE3) at 18°C from pGEX-4T-1 (GE Healthcare), and hexa-histidine tagged mouse importin-α2 (SwissProt entry P52293) comprising residues 70–498 (ImpA) was overexpressed separately from the same strain at 18°C from pET-28a.

Article Title: ESCRT-III mediates budding across the inner nuclear membrane and regulates its integrity
Article Snippet: Plasmids pcDNA3.1-UL34 and pcDNA3.1-UL31 used for the expression of HSV-1 UL34 and UL31, respectively, were reported previously . pCHMP4B-EGFP encoding a fusion protein of CHMP4B and EGFP (CHMP4B-EGFP) were constructed by cloning CHMP4B cDNA, amplified by PCR from pCMV(Δ5)-CHMP4B , (kindly provided by W. I. Sundquist), into pEGFP-N1 (Clontech, Mountain View, CA) in frame with EGFP. .. To generate fusion proteins of GST with the ALIX Bro domain (amino acids 1–358) and the ALIX V domain (amino acids 362–702) (GST-ALIX-Bro and GST-ALIX-V, respectively), pGEX-ALIX-Bro and pGEX-ALIX-V were constructed by cloning the appropriate ALI X domain, amplified by PCR from pCI-FLAG-ALIX, and cloned into pGEX-4T-1 (GE Healthcare Bio-Sciences, Piscataway, NJ).

Article Title: Human babesiosis: Indication of a molecular mimicry between thrombospondin domains from a novel Babesia microti BmP53 protein and host platelets molecules
Article Snippet: For cloning, two pairs of oligonucleotide primers with Bam HI and Xho I restriction enzyme sites were designed and used for the amplification of both truncates, BmP53tr1-TSP1 (forward primer: 5′-CGGGATCCGAGAAGATAAAGGATGAA-3′ ; reverse primer: 5′-CGCTCGAGTTATTTATAGCTAGAGCTCAG -3′ ) and BmP53tr2 (forward primer: 5′-CGGGATCCAGGTTAATGAAGCTGCCA-3′ ; reverse primer: 5′-CGCTCGAGTTACGCAAATTGGGCAAATGA-3′ ). .. The PCR products were ligated to the Bam HI and Xho I restriction sites of the glutathione S-transferase (GST) fusion expression vector, pGEX-4T-1 (GE Healthcare Life Sciences, UK). .. The constructs of the resulting plasmids were checked for accurate insertion by digestion with Bam HI and Xho I according to the manufacturer’s instructions (ROCHE Diagnostics, Germany).

Article Title: Transcriptional regulation of autophagy-lysosomal function in BRAF-driven melanoma progression and chemoresistance
Article Snippet: Flag-tagged BRAFV600E was cloned into pCDH-CMV-MCS-EF1-puro backbone (System Biosciences; SBI) to generate lentiviral transfer constructs. shRNA-resistant TFEB mutants were generated using site-directed mutagenesis (Q5 0552S, New England Biolab). .. For expression of GST fusion in Escherichia coli BL21 cells, the full-length TFEB, pro-TGFβ, and its point mutants were cloned into the pGEX-4T-1 (Pharmacia Amersham) vector or the pET32a (EMD Biosciences) vector. .. The plasmids of ZKSCAN3-V5 was provided by Dr. Pinghui Feng, University of Southern California, USA.

Article Title: Precision Targeting of BFL-1/A1 and an ATM Co-dependency in Human Cancer
Article Snippet: Protein purity and identity was confirmed by Coomassie staining and western blot analysis using a mouse monoclonal anti-His6 tag antibody (Abcam). .. Recombinant anti-apoptotic BCL-XL ΔC (aa 1–212) and MCL-1ΔNΔC (aa 172–329) were cloned into the pGEX-4T-1 (GE Healthcare, N-terminal GST tag) expression vector, expressed in BL21(DE3) Escherichia coli (Sigma Aldrich), and purified as described previously ( ). .. Protein expression was induced using 0.5 mM IPTG overnight at 37°C.

Western Blot:

Article Title: Precision Targeting of BFL-1/A1 and an ATM Co-dependency in Human Cancer
Article Snippet: Protein purity and identity was confirmed by Coomassie staining and western blot analysis using a mouse monoclonal anti-His6 tag antibody (Abcam). .. Recombinant anti-apoptotic BCL-XL ΔC (aa 1–212) and MCL-1ΔNΔC (aa 172–329) were cloned into the pGEX-4T-1 (GE Healthcare, N-terminal GST tag) expression vector, expressed in BL21(DE3) Escherichia coli (Sigma Aldrich), and purified as described previously ( ).

Transformation Assay:

Article Title: Recombinant Paracoccin Reproduces the Biological Properties of the Native Protein and Induces Protective Th1 Immunity against Paracoccidioides brasiliensis Infection
Article Snippet: First, the largest exon was amplified from P. brasiliensis genomic DNA using the oligonucleotide primers FPADG ( 5′-CTGGATCCATGCAAGCACCCGACCAAC-3′ ) and RPADG ( 5′-CGGGAATTCCTACCAACTCGTTATTGATAGAGCGATAA-3′ ), and then cloned into pGEX-4T-1 (GE Healthcare, Upsala, Sweden) flanked by Bam HI and Eco RI restriction sites. .. In the first strategy, the paracoccin fragment, referred to as rPCNexon4 , is fused to a GST tag in the pGEX vector, whereas in the second strategy, the 5′-UTR region, which drives transcription of paracoccin (rPCNfull ) was also synthesized.

Article Title: High yield production of pigeon circovirus capsid protein in the E. coli by evaluating the key parameters needed for protein expression
Article Snippet: This cDNA was cloned into either pET28a, pET32a (Novagen, Madison, WI) or pGEX-4T-1 (GE Healthcare, Piscataway, NJ) individually using Eco R1 and Xho I (Takara, Japan) restriction sites. .. To improve the codon usage of the cap gene from PiCV, a second cDNA sequence was synthesized by Genemark Biosci & Tech Co that contained the codons that were optimized for E. coli ; this was also ligated individually into the same three E. coli expression vectors using the same restriction sites; these constructs were designated pHis-Capopt , pTrx-His-Capopt and pGST-Capopt , respectively (Figure , panel b, f and d).

Article Title: Bioengineered bacterial vesicles as biological nano-heaters for optoacoustic imaging
Article Snippet: Plasmid construction, bacterial strain and growth The melA gene of Rhizobium etli encoding tyrosinase (kindly provided by Professor Guillermo Gosset, Universidad Nacional Autónoma de México, Mexico) was cloned into pGEX-4T-1 (GE Healthcare, Freiburg, Germany). .. Plasmid construction, bacterial strain and growth The melA gene of Rhizobium etli encoding tyrosinase (kindly provided by Professor Guillermo Gosset, Universidad Nacional Autónoma de México, Mexico) was cloned into pGEX-4T-1 (GE Healthcare, Freiburg, Germany).

Article Title: Analysis of Metal-Binding Features of the Wild Type and Two Domain-Truncated Mutant Variants of Littorina littorea Metallothionein Reveals Its Cd-Specific Character
Article Snippet: After PCR amplification (35 cycles: 95 °C 30 s, 50 °C 30 s, and 72 °C 30 s, using Expand High Fidelity (Roche Diagnostics S.L:, San Cugat del Vallès, Spain) thermostable DNA polymerase) of the synthetic cDNAs using the flanking primers 5′-TTTATTGGATCCATGAGCTC-3′ (LlwtMT forward); 5′-TTTCTCGAGTCATTTGCATG-3′ (LlwtMT reverse); 5′-TTTATTGGATCCGGTAAGGG-3′ (Lltr1MT forward); 5′-TTTCTCGAGTTACTTACAGG-3′ (Lltr1MT reverse); 5′-TTTATTGGATCCGGCAAAGGG-3′ (Lltr2MT forward); and 5′-TTTCTCGAGTTATTTGCAAG-3′ (Lltr2MT reverse), they were subsequently digested with BamHI and XhoI restriction enzymes, and the resulting products were ligated in-frame (DNA ligation kit, Takara Bio, Kusatsu, Shiga, Japan) in the pGEX-4T-1 (Amersham-GE Healthcare Europe, Cerdanyola del Valles, Spain) E. coli expression vector, which yields GST-fusion proteins. .. The E. coli MachI strain was used for cloning and sequencing.

Article Title: Does Variation of the Inter-Domain Linker Sequence Modulate the Metal Binding Behaviour of Helix pomatia Cd-Metallothionein?
Article Snippet: They were subsequently digested with BamH I and Xho I restriction enzymes, and the resulting products were ligated in-frame (DNA ligation kit, Takara Bio, Kusatsu, Shiga, Japan) in the pGEX-4T-1 (Amersham-GE Healthcare Europe, Cerdanyola del Valles, Spain) E. coli expression vector, which yields GST-fusion proteins. .. The E. coli MachI strain was used for cloning and sequencing.

Article Title: Plasmodium falciparum ookinete expression of plasmepsin VII and plasmepsin X
Article Snippet: PCR products were gel purified on a 0.8 % agarose gel using the PureLink gel extraction kit (Invitrogen, Carlsbad, CA, USA), ligated into pCR4-TOPO (Invitrogen, Carlsbad, CA, USA), transformed into Top10 competent cells (Invitrogen, Carlsbad, CA, USA) and sequence-verified (Eton Bioscience, San Diego, CA, USA). .. PfPM VII and PfPM X genes were then cloned into expression vectors pET32 and pGEX 4T-1 (GE Healthcare, Piscataway, NJ, USA), respectively.

Crystallization Assay:

Article Title: Structural basis for recruiting and shuttling of the spliceosomal deubiquitinase USP4 by SART3
Article Snippet: Final protein complex was concentrated to 20 mg ml−1 in same buffer using Vivaspin concentrator (Vivagen) for crystallization trials. .. The glutathione S-transferase (GST)-tagged SART3 NLS, comprising residues 601–649, was overexpressed in E. coli strain Rossetta 2(DE3) at 18°C from pGEX-4T-1 (GE Healthcare), and hexa-histidine tagged mouse importin-α2 (SwissProt entry P52293) comprising residues 70–498 (ImpA) was overexpressed separately from the same strain at 18°C from pET-28a.

Flow Cytometry:

Article Title: C3G forms complexes with Bcr-Abl and p38? MAPK at the focal adhesions in chronic myeloid leukemia cells: implication in the regulation of leukemic cell adhesion
Article Snippet: To do so, fragments were amplified by PCR and cloned into Eco RI-Xho I sites of pGEX-4T-1 (GE Healthcare Life Sciences). .. GST-Abl-SH3 and GST-C3G-SH3-b constructs have been described previously [ ].

Chromatography:

Article Title: Precision Targeting of BFL-1/A1 and an ATM Co-dependency in Human Cancer
Article Snippet: Recombinant anti-apoptotic BCL-XL ΔC (aa 1–212) and MCL-1ΔNΔC (aa 172–329) were cloned into the pGEX-4T-1 (GE Healthcare, N-terminal GST tag) expression vector, expressed in BL21(DE3) Escherichia coli (Sigma Aldrich), and purified as described previously ( ). .. Protein expression was induced using 0.5 mM IPTG overnight at 37°C.

Concentration Assay:

Article Title: Bioengineered bacterial vesicles as biological nano-heaters for optoacoustic imaging
Article Snippet: Plasmid construction, bacterial strain and growth The melA gene of Rhizobium etli encoding tyrosinase (kindly provided by Professor Guillermo Gosset, Universidad Nacional Autónoma de México, Mexico) was cloned into pGEX-4T-1 (GE Healthcare, Freiburg, Germany). .. The construct pGEX-4T-1-melA was transformed into msbB mutant W3110-K12 E. coli (kindly provided by Professor Sangyong Jon, KAIST, South Korea) .

Generated:

Article Title: Transcriptional regulation of autophagy-lysosomal function in BRAF-driven melanoma progression and chemoresistance
Article Snippet: Flag-tagged BRAFV600E was cloned into pCDH-CMV-MCS-EF1-puro backbone (System Biosciences; SBI) to generate lentiviral transfer constructs. shRNA-resistant TFEB mutants were generated using site-directed mutagenesis (Q5 0552S, New England Biolab). .. For expression of GST fusion in Escherichia coli BL21 cells, the full-length TFEB, pro-TGFβ, and its point mutants were cloned into the pGEX-4T-1 (Pharmacia Amersham) vector or the pET32a (EMD Biosciences) vector.

DNA Sequencing:

Article Title: Recombinant Paracoccin Reproduces the Biological Properties of the Native Protein and Induces Protective Th1 Immunity against Paracoccidioides brasiliensis Infection
Article Snippet: First, the largest exon was amplified from P. brasiliensis genomic DNA using the oligonucleotide primers FPADG ( 5′-CTGGATCCATGCAAGCACCCGACCAAC-3′ ) and RPADG ( 5′-CGGGAATTCCTACCAACTCGTTATTGATAGAGCGATAA-3′ ), and then cloned into pGEX-4T-1 (GE Healthcare, Upsala, Sweden) flanked by Bam HI and Eco RI restriction sites. .. Competent E. coli BL21 (DE3) cells were transformed with the recombinant vectors, and clones resistant to ampicillin were screened for the presence of the inserts by PCR.

Article Title: Nuclear GSK3β promotes tumorigenesis by phosphorylating KDM1A and inducing its deubiquitination by USP22
Article Snippet: GST-tagged KDM1A or KDM1A S683A/S687A was constructed by cloning the wild-type or mutant cDNA into pGEX-4T-1 (GE Healthcare). .. The cDNAs of three CK1 isoforms were amplified and cloned into pcDNA3-HA vector.

Sequencing:

Article Title: High yield production of pigeon circovirus capsid protein in the E. coli by evaluating the key parameters needed for protein expression
Article Snippet: A 822 bp of cDNA fragment consisting of the cap gene that encodes the full-length PiCV capsid protein was synthesized by Genemark Biosci & Tech Co. (Taichung, Taiwan) based on the published sequence (Columbid circovirus, isolate 9030; Accession No. AJ298229). .. This cDNA was cloned into either pET28a, pET32a (Novagen, Madison, WI) or pGEX-4T-1 (GE Healthcare, Piscataway, NJ) individually using Eco R1 and Xho I (Takara, Japan) restriction sites.

Article Title: REGULATION OF PROGESTERONE RECEPTOR ACTIVITY BY CYCLIN DEPENDENT KINASES 1 AND 2 OCCURS IN PART BY PHOSPHORYLATION OF THE SRC-1 CARBOXYL-TERMINUS
Article Snippet: Eight fragments of SRC-1 (F1–F8) were amplified by PCR from the full length wild type (wt) pCR3.1-SRC-1 construct using Platinum Pfx polymerase (Invitrogen) and primers indicated in , with incorporation of BamHI and NotI restriction sites at the 5’ and 3’ ends, respectively. .. PCR products were ligated in frame into pGEX-4T-1 (GE Healthcare) and clones were verified by sequencing. .. Constructs were transformed into E. coli BL21 (DE3) cells (Stratagene, La Jolla, CA) and fusion protein expression was induced with 1 mM IPTG.

Article Title: Analysis of Metal-Binding Features of the Wild Type and Two Domain-Truncated Mutant Variants of Littorina littorea Metallothionein Reveals Its Cd-Specific Character
Article Snippet: For the construction of the pGEX plasmids containing the wild type sequence of Littorina littorea or the mutants, the cDNAs encoding for these sequences were designed on the basis of the flanking regions in wild-type LlwtMT cDNA, and to encode the truncated sequences in the corresponding Lltr1MT and Lltr2MT cDNAs, taking out nucleotides 4 to 111 and 16 to 111, respectively. .. After PCR amplification (35 cycles: 95 °C 30 s, 50 °C 30 s, and 72 °C 30 s, using Expand High Fidelity (Roche Diagnostics S.L:, San Cugat del Vallès, Spain) thermostable DNA polymerase) of the synthetic cDNAs using the flanking primers 5′-TTTATTGGATCCATGAGCTC-3′ (LlwtMT forward); 5′-TTTCTCGAGTCATTTGCATG-3′ (LlwtMT reverse); 5′-TTTATTGGATCCGGTAAGGG-3′ (Lltr1MT forward); 5′-TTTCTCGAGTTACTTACAGG-3′ (Lltr1MT reverse); 5′-TTTATTGGATCCGGCAAAGGG-3′ (Lltr2MT forward); and 5′-TTTCTCGAGTTATTTGCAAG-3′ (Lltr2MT reverse), they were subsequently digested with BamHI and XhoI restriction enzymes, and the resulting products were ligated in-frame (DNA ligation kit, Takara Bio, Kusatsu, Shiga, Japan) in the pGEX-4T-1 (Amersham-GE Healthcare Europe, Cerdanyola del Valles, Spain) E. coli expression vector, which yields GST-fusion proteins.

Article Title: Does Variation of the Inter-Domain Linker Sequence Modulate the Metal Binding Behaviour of Helix pomatia Cd-Metallothionein?
Article Snippet: Two HpCdMT mutants were designed to replace the only two residues (-KT-) that act as a linker between the two nine-Cys domains in the HpCdMT sequence with the corresponding linkers from the (giant keyhole limpet) M. crenulata MT [ ] (-VKTEAKTT-; HpCdMcMT protein) or from wheat (T. aestivum ) Ec-1 MT [ ] (-SARSGAAA-; HpCdPlMT protein) ( ). .. They were subsequently digested with BamH I and Xho I restriction enzymes, and the resulting products were ligated in-frame (DNA ligation kit, Takara Bio, Kusatsu, Shiga, Japan) in the pGEX-4T-1 (Amersham-GE Healthcare Europe, Cerdanyola del Valles, Spain) E. coli expression vector, which yields GST-fusion proteins.

Article Title: Nuclear GSK3β promotes tumorigenesis by phosphorylating KDM1A and inducing its deubiquitination by USP22
Article Snippet: GST-tagged KDM1A or KDM1A S683A/S687A was constructed by cloning the wild-type or mutant cDNA into pGEX-4T-1 (GE Healthcare). .. The cDNAs of three CK1 isoforms were amplified and cloned into pcDNA3-HA vector.

Article Title: Transcriptional regulation of autophagy-lysosomal function in BRAF-driven melanoma progression and chemoresistance
Article Snippet: For expression of GST fusion in Escherichia coli BL21 cells, the full-length TFEB, pro-TGFβ, and its point mutants were cloned into the pGEX-4T-1 (Pharmacia Amersham) vector or the pET32a (EMD Biosciences) vector. .. For expression of GST fusion in Escherichia coli BL21 cells, the full-length TFEB, pro-TGFβ, and its point mutants were cloned into the pGEX-4T-1 (Pharmacia Amersham) vector or the pET32a (EMD Biosciences) vector.

Sonication:

Article Title: Polarized Exocytosis Induces Compensatory Endocytosis by Sec4p-Regulated Cortical Actin Polymerization
Article Snippet: For purification of GST-fusion proteins from bacterial extracts, and their subsequent use in actin polymerization assays, coding sequences for SLA1 , LAS17 , SEC4 , and YPT1 were sub-cloned into Bam HI and Sal I sites in pGEX-4T-1 (GE Healthcare Life Sciences, Pittsburg, PA). .. To induce GST-fusion protein expression, 250 ml of early log-phase cultures grown in LB at 37°C were treated with 1 mM IPTG and, after an additional 2 h of growth at 23°C, cells were pelleted by centrifugation at 5,000 x g for 30 min at 4°C.

Article Title: Full length RTN3 regulates turnover of tubular endoplasmic reticulum via selective autophagy
Article Snippet: LC3s, GABARAPs and Ub proteins were cloned, as GST fusion proteins, into pGEX-4T-1 (GE Healthcare; Little Chalfont, UK) and expressed in Escherichia coli BL21 (DE3) cells in LB medium. .. Expression was induced by addition of 0.5 mM IPTG and cells were incubated at 37°C for 5 hr.

Affinity Purification:

Article Title: Polarized Exocytosis Induces Compensatory Endocytosis by Sec4p-Regulated Cortical Actin Polymerization
Article Snippet: Paragraph title: In Vitro Pyrene-Actin Polymerization Assay and Affinity Purification of Fusion Proteins ... For purification of GST-fusion proteins from bacterial extracts, and their subsequent use in actin polymerization assays, coding sequences for SLA1 , LAS17 , SEC4 , and YPT1 were sub-cloned into Bam HI and Sal I sites in pGEX-4T-1 (GE Healthcare Life Sciences, Pittsburg, PA).

Binding Assay:

Article Title: Analysis of Metal-Binding Features of the Wild Type and Two Domain-Truncated Mutant Variants of Littorina littorea Metallothionein Reveals Its Cd-Specific Character
Article Snippet: Two LltrMT truncation mutants (LltrMT1 and LltrMT2) were designed to lack the first metal binding domain completely. .. After PCR amplification (35 cycles: 95 °C 30 s, 50 °C 30 s, and 72 °C 30 s, using Expand High Fidelity (Roche Diagnostics S.L:, San Cugat del Vallès, Spain) thermostable DNA polymerase) of the synthetic cDNAs using the flanking primers 5′-TTTATTGGATCCATGAGCTC-3′ (LlwtMT forward); 5′-TTTCTCGAGTCATTTGCATG-3′ (LlwtMT reverse); 5′-TTTATTGGATCCGGTAAGGG-3′ (Lltr1MT forward); 5′-TTTCTCGAGTTACTTACAGG-3′ (Lltr1MT reverse); 5′-TTTATTGGATCCGGCAAAGGG-3′ (Lltr2MT forward); and 5′-TTTCTCGAGTTATTTGCAAG-3′ (Lltr2MT reverse), they were subsequently digested with BamHI and XhoI restriction enzymes, and the resulting products were ligated in-frame (DNA ligation kit, Takara Bio, Kusatsu, Shiga, Japan) in the pGEX-4T-1 (Amersham-GE Healthcare Europe, Cerdanyola del Valles, Spain) E. coli expression vector, which yields GST-fusion proteins.

Article Title: CIB1 is a Regulator of Pathological Cardiac Hypertrophy
Article Snippet: To generate GST fusion proteins, CIB1 cDNA sequences were amplified by PCR and cloned into pGEX-4T-1 (Amersham Pharmacia Biotech). .. To generate GST fusion proteins, CIB1 cDNA sequences were amplified by PCR and cloned into pGEX-4T-1 (Amersham Pharmacia Biotech).

Article Title: Polarized Exocytosis Induces Compensatory Endocytosis by Sec4p-Regulated Cortical Actin Polymerization
Article Snippet: For purification of GST-fusion proteins from bacterial extracts, and their subsequent use in actin polymerization assays, coding sequences for SLA1 , LAS17 , SEC4 , and YPT1 were sub-cloned into Bam HI and Sal I sites in pGEX-4T-1 (GE Healthcare Life Sciences, Pittsburg, PA). .. To induce GST-fusion protein expression, 250 ml of early log-phase cultures grown in LB at 37°C were treated with 1 mM IPTG and, after an additional 2 h of growth at 23°C, cells were pelleted by centrifugation at 5,000 x g for 30 min at 4°C.

Magnetic Beads:

Article Title: Polarized Exocytosis Induces Compensatory Endocytosis by Sec4p-Regulated Cortical Actin Polymerization
Article Snippet: For purification of GST-fusion proteins from bacterial extracts, and their subsequent use in actin polymerization assays, coding sequences for SLA1 , LAS17 , SEC4 , and YPT1 were sub-cloned into Bam HI and Sal I sites in pGEX-4T-1 (GE Healthcare Life Sciences, Pittsburg, PA). .. After resuspension in 20 ml binding buffer (125 mM Tris pH 8.0, 150 mM NaCl) containing EDTA-free protease inhibitors (Roche, Basel, CH), cells were lysed by sonication using a digital sonifier (Branson Ultrasonics Co., Danbury, CT) at 20% amplitude for 3 min with cycle bursts of 1 s on and 3 s off.

Mutagenesis:

Article Title: F-Box Protein FBXO31 Mediates Cyclin D1 Degradation to Induce G1 Arrest Following DNA Damage
Article Snippet: The plasmid pLNCX2-FBXO31 was used as a template to generate FBXO31 mutants using the QuikChange XL site-directed mutagenesis kit (Stratagene). .. To generate in-frame GST-tagged fusion proteins, the wild type and mutant FBXO31 sequences were PCR amplified (using primers WT1_forward 5’-CGGGATCCGCTGCCACAGTGGAGTGC-3’ and WT1_reverse 5’-GGAATTCGATGAGGTCGTCGGGGCG-3’, or WT2_forward 5’-CGGAATTCCGG ATCCAGCTGCCCGAC-3’ and WT2_reverse 5’-ACGTCGACCACGGGCAGCACG AACGG-3’), digested with BamH1 and EcoR1, and cloned into pGEX-4T-1 (Amersham). .. In vitro ATM kinase assays were performed as described previously .

Article Title: Bioengineered bacterial vesicles as biological nano-heaters for optoacoustic imaging
Article Snippet: Plasmid construction, bacterial strain and growth The melA gene of Rhizobium etli encoding tyrosinase (kindly provided by Professor Guillermo Gosset, Universidad Nacional Autónoma de México, Mexico) was cloned into pGEX-4T-1 (GE Healthcare, Freiburg, Germany). .. Plasmid construction, bacterial strain and growth The melA gene of Rhizobium etli encoding tyrosinase (kindly provided by Professor Guillermo Gosset, Universidad Nacional Autónoma de México, Mexico) was cloned into pGEX-4T-1 (GE Healthcare, Freiburg, Germany).

Article Title: Nuclear GSK3β promotes tumorigenesis by phosphorylating KDM1A and inducing its deubiquitination by USP22
Article Snippet: Site-directed mutagenesis in KDM1A was introduced using the QuikChange site-directed mutagenesis kit (Agilent Technologies). .. GST-tagged KDM1A or KDM1A S683A/S687A was constructed by cloning the wild-type or mutant cDNA into pGEX-4T-1 (GE Healthcare). .. Lentiviral expression of KDM1A-shR, KDM1A S683D, or KDM1A S683A was achieved by cloning the cDNA into a pLVX-IRES-Hyg vector.

Article Title: Transcriptional regulation of autophagy-lysosomal function in BRAF-driven melanoma progression and chemoresistance
Article Snippet: Flag-tagged BRAFV600E was cloned into pCDH-CMV-MCS-EF1-puro backbone (System Biosciences; SBI) to generate lentiviral transfer constructs. shRNA-resistant TFEB mutants were generated using site-directed mutagenesis (Q5 0552S, New England Biolab). .. For expression of GST fusion in Escherichia coli BL21 cells, the full-length TFEB, pro-TGFβ, and its point mutants were cloned into the pGEX-4T-1 (Pharmacia Amersham) vector or the pET32a (EMD Biosciences) vector.

Transfection:

Article Title: Full length RTN3 regulates turnover of tubular endoplasmic reticulum via selective autophagy
Article Snippet: LC3s, GABARAPs and Ub proteins were cloned, as GST fusion proteins, into pGEX-4T-1 (GE Healthcare; Little Chalfont, UK) and expressed in Escherichia coli BL21 (DE3) cells in LB medium. .. LC3s, GABARAPs and Ub proteins were cloned, as GST fusion proteins, into pGEX-4T-1 (GE Healthcare; Little Chalfont, UK) and expressed in Escherichia coli BL21 (DE3) cells in LB medium.

Purification:

Article Title: REGULATION OF PROGESTERONE RECEPTOR ACTIVITY BY CYCLIN DEPENDENT KINASES 1 AND 2 OCCURS IN PART BY PHOSPHORYLATION OF THE SRC-1 CARBOXYL-TERMINUS
Article Snippet: PCR products were ligated in frame into pGEX-4T-1 (GE Healthcare) and clones were verified by sequencing. .. Constructs were transformed into E. coli BL21 (DE3) cells (Stratagene, La Jolla, CA) and fusion protein expression was induced with 1 mM IPTG.

Article Title: Plasmodium falciparum ookinete expression of plasmepsin VII and plasmepsin X
Article Snippet: PCR products were gel purified on a 0.8 % agarose gel using the PureLink gel extraction kit (Invitrogen, Carlsbad, CA, USA), ligated into pCR4-TOPO (Invitrogen, Carlsbad, CA, USA), transformed into Top10 competent cells (Invitrogen, Carlsbad, CA, USA) and sequence-verified (Eton Bioscience, San Diego, CA, USA). .. PfPM VII and PfPM X genes were then cloned into expression vectors pET32 and pGEX 4T-1 (GE Healthcare, Piscataway, NJ, USA), respectively.

Article Title: Polarized Exocytosis Induces Compensatory Endocytosis by Sec4p-Regulated Cortical Actin Polymerization
Article Snippet: Bgl2p polarized exocytosis was assayed as previously described [ ]. .. For purification of GST-fusion proteins from bacterial extracts, and their subsequent use in actin polymerization assays, coding sequences for SLA1 , LAS17 , SEC4 , and YPT1 were sub-cloned into Bam HI and Sal I sites in pGEX-4T-1 (GE Healthcare Life Sciences, Pittsburg, PA). .. For protein expression, plasmids were transformed into the Escherichia coli strain BL21 (DE3) RIL.

Article Title: Structural basis for recruiting and shuttling of the spliceosomal deubiquitinase USP4 by SART3
Article Snippet: Paragraph title: Protein expression and purification ... The glutathione S-transferase (GST)-tagged SART3 NLS, comprising residues 601–649, was overexpressed in E. coli strain Rossetta 2(DE3) at 18°C from pGEX-4T-1 (GE Healthcare), and hexa-histidine tagged mouse importin-α2 (SwissProt entry P52293) comprising residues 70–498 (ImpA) was overexpressed separately from the same strain at 18°C from pET-28a.

Article Title: Precision Targeting of BFL-1/A1 and an ATM Co-dependency in Human Cancer
Article Snippet: Protein purity and identity was confirmed by Coomassie staining and western blot analysis using a mouse monoclonal anti-His6 tag antibody (Abcam). .. Recombinant anti-apoptotic BCL-XL ΔC (aa 1–212) and MCL-1ΔNΔC (aa 172–329) were cloned into the pGEX-4T-1 (GE Healthcare, N-terminal GST tag) expression vector, expressed in BL21(DE3) Escherichia coli (Sigma Aldrich), and purified as described previously ( ). .. Protein expression was induced using 0.5 mM IPTG overnight at 37°C.

Polymerase Chain Reaction:

Article Title: Recombinant Paracoccin Reproduces the Biological Properties of the Native Protein and Induces Protective Th1 Immunity against Paracoccidioides brasiliensis Infection
Article Snippet: First, the largest exon was amplified from P. brasiliensis genomic DNA using the oligonucleotide primers FPADG ( 5′-CTGGATCCATGCAAGCACCCGACCAAC-3′ ) and RPADG ( 5′-CGGGAATTCCTACCAACTCGTTATTGATAGAGCGATAA-3′ ), and then cloned into pGEX-4T-1 (GE Healthcare, Upsala, Sweden) flanked by Bam HI and Eco RI restriction sites. .. In the first strategy, the paracoccin fragment, referred to as rPCNexon4 , is fused to a GST tag in the pGEX vector, whereas in the second strategy, the 5′-UTR region, which drives transcription of paracoccin (rPCNfull ) was also synthesized.

Article Title: C3G forms complexes with Bcr-Abl and p38? MAPK at the focal adhesions in chronic myeloid leukemia cells: implication in the regulation of leukemic cell adhesion
Article Snippet: Constructs: Abi1-SH3, Abi1-SH3-b (SH3-binding), Cbl-SH3-b and p130Cas-SH3, p130Cas-P1 (proline-rich domain 1 or SH3-b1) and p130Cas-P2 (SH3-b2) domains were expressed as GST-fusion proteins. .. To do so, fragments were amplified by PCR and cloned into Eco RI-Xho I sites of pGEX-4T-1 (GE Healthcare Life Sciences). .. The oligos used were Abi1SH3-F: 5´-GGGGAATTCCCCAAGAATTATATTGAGAAAGTT-3´ and Abi1SH3-R: 5´-GGGCTCGAGTTAATCAGTATAGTGCATGATTGA-3; Abi1SH3b-F: 5´-GGGGAATTCCCCATTGCTGTGCCTACA-3´ and Abi1SH3b-R: 5´-GGGCTCGAGCAGCCTCCTCATCTTCAT-3´; CblSH3b-F: 5´-GAGGAATTCCCGCCTTCTCCATTCTCC-3´ and CblSH3b-R: 5´-GGGCTCGAGAGGTGGCAGTTTTGGCAC-3´; p130CasSH3-F: 5´-AGGGAATTCAACCACCTGAACGTGCTG-3´ and p130CasSH3-R: 5´-AGGCTCGAGGCCCACCAAGATCTTGAG-3; p130CasP1-F: 5´-AGGGAATTCGATAAGAAGCCAGCAGGG-3 and p130CasP1-R: 5´-AGGCTCGAGGTAGACGCTGTCTGGCTG-3´; p130CasP2-F: 5´-AGGGAATTCTCACTGCTCTTCAGACGG-3´ and p130CasP2-R: 5´-GGGCTCGAGGGTGAACTTAGGGGGTGA-3´.

Article Title: REGULATION OF PROGESTERONE RECEPTOR ACTIVITY BY CYCLIN DEPENDENT KINASES 1 AND 2 OCCURS IN PART BY PHOSPHORYLATION OF THE SRC-1 CARBOXYL-TERMINUS
Article Snippet: Eight fragments of SRC-1 (F1–F8) were amplified by PCR from the full length wild type (wt) pCR3.1-SRC-1 construct using Platinum Pfx polymerase (Invitrogen) and primers indicated in , with incorporation of BamHI and NotI restriction sites at the 5’ and 3’ ends, respectively. .. PCR products were ligated in frame into pGEX-4T-1 (GE Healthcare) and clones were verified by sequencing. .. Constructs were transformed into E. coli BL21 (DE3) cells (Stratagene, La Jolla, CA) and fusion protein expression was induced with 1 mM IPTG.

Article Title: F-Box Protein FBXO31 Mediates Cyclin D1 Degradation to Induce G1 Arrest Following DNA Damage
Article Snippet: The plasmid pLNCX2-FBXO31 was used as a template to generate FBXO31 mutants using the QuikChange XL site-directed mutagenesis kit (Stratagene). .. To generate in-frame GST-tagged fusion proteins, the wild type and mutant FBXO31 sequences were PCR amplified (using primers WT1_forward 5’-CGGGATCCGCTGCCACAGTGGAGTGC-3’ and WT1_reverse 5’-GGAATTCGATGAGGTCGTCGGGGCG-3’, or WT2_forward 5’-CGGAATTCCGG ATCCAGCTGCCCGAC-3’ and WT2_reverse 5’-ACGTCGACCACGGGCAGCACG AACGG-3’), digested with BamH1 and EcoR1, and cloned into pGEX-4T-1 (Amersham). .. In vitro ATM kinase assays were performed as described previously .

Article Title: Analysis of Metal-Binding Features of the Wild Type and Two Domain-Truncated Mutant Variants of Littorina littorea Metallothionein Reveals Its Cd-Specific Character
Article Snippet: The LlwtMT, Lltr1MT, and Lltr2MT coding sequences designed this way were purchased as synthetic DNAs IDT & Conda Labs (Madrid, Spain). .. After PCR amplification (35 cycles: 95 °C 30 s, 50 °C 30 s, and 72 °C 30 s, using Expand High Fidelity (Roche Diagnostics S.L:, San Cugat del Vallès, Spain) thermostable DNA polymerase) of the synthetic cDNAs using the flanking primers 5′-TTTATTGGATCCATGAGCTC-3′ (LlwtMT forward); 5′-TTTCTCGAGTCATTTGCATG-3′ (LlwtMT reverse); 5′-TTTATTGGATCCGGTAAGGG-3′ (Lltr1MT forward); 5′-TTTCTCGAGTTACTTACAGG-3′ (Lltr1MT reverse); 5′-TTTATTGGATCCGGCAAAGGG-3′ (Lltr2MT forward); and 5′-TTTCTCGAGTTATTTGCAAG-3′ (Lltr2MT reverse), they were subsequently digested with BamHI and XhoI restriction enzymes, and the resulting products were ligated in-frame (DNA ligation kit, Takara Bio, Kusatsu, Shiga, Japan) in the pGEX-4T-1 (Amersham-GE Healthcare Europe, Cerdanyola del Valles, Spain) E. coli expression vector, which yields GST-fusion proteins. .. DNA sequencing allowed confirming of all the DNA constructs (ABIPRISM 310, Applied Biosystems, Foster City, CA, USA) using the BigDye Terminator.

Article Title: CIB1 is a Regulator of Pathological Cardiac Hypertrophy
Article Snippet: Cotransformation of pGBKT7-CnBshort and pGAD-CnA served as a positive control. .. To generate GST fusion proteins, CIB1 cDNA sequences were amplified by PCR and cloned into pGEX-4T-1 (Amersham Pharmacia Biotech). .. The fusion proteins were expressed in E. coli BL21 cells (Invitrogen) and precipitated with glutathione-sepharose beads (GE Healthcare).

Article Title: Does Variation of the Inter-Domain Linker Sequence Modulate the Metal Binding Behaviour of Helix pomatia Cd-Metallothionein?
Article Snippet: After PCR amplification (35 cycles: 95 °C 30 s, 50 °C 30 s, and 72 °C 30 s, using Expand High Fidelity (Roche) thermostable DNA polymerase) of the synthetic cDNAs using the flanking primers: 5’-TTTATTGGATCCGGTAAGGG-3’ (HpCdMcMT forward); 5’-TTTCTCGAGTTACTTACAGG-3’ (HpCdMcMT reverse); 5’-TTTATTGGATCCGGCAAAGGG-3’ (HpCdPlMT forward); and 5’-TTTCTCGAGTTATTTGCAAG-3’ (HpCdPlMT reverse). .. They were subsequently digested with BamH I and Xho I restriction enzymes, and the resulting products were ligated in-frame (DNA ligation kit, Takara Bio, Kusatsu, Shiga, Japan) in the pGEX-4T-1 (Amersham-GE Healthcare Europe, Cerdanyola del Valles, Spain) E. coli expression vector, which yields GST-fusion proteins.

Article Title: Plasmodium falciparum ookinete expression of plasmepsin VII and plasmepsin X
Article Snippet: PCR products were gel purified on a 0.8 % agarose gel using the PureLink gel extraction kit (Invitrogen, Carlsbad, CA, USA), ligated into pCR4-TOPO (Invitrogen, Carlsbad, CA, USA), transformed into Top10 competent cells (Invitrogen, Carlsbad, CA, USA) and sequence-verified (Eton Bioscience, San Diego, CA, USA). .. PfPM VII and PfPM X genes were then cloned into expression vectors pET32 and pGEX 4T-1 (GE Healthcare, Piscataway, NJ, USA), respectively.

Article Title: ESCRT-III mediates budding across the inner nuclear membrane and regulates its integrity
Article Snippet: ALIX cDNA was cloned into pcDNA3.1( + ) (Invitrogen, Carlsbad, CA) in frame with a Flag-epitope to generate pcDNA3.1-Flag-ALIX. pcDNA3.1-Flag-ALIX-I212D was constructed by replacing ALIX codons I212 in pcDNA3.1-Flag-ALIX with aspartic acid codons as reported previously . .. To generate fusion proteins of GST with the ALIX Bro domain (amino acids 1–358) and the ALIX V domain (amino acids 362–702) (GST-ALIX-Bro and GST-ALIX-V, respectively), pGEX-ALIX-Bro and pGEX-ALIX-V were constructed by cloning the appropriate ALI X domain, amplified by PCR from pCI-FLAG-ALIX, and cloned into pGEX-4T-1 (GE Healthcare Bio-Sciences, Piscataway, NJ). .. Sense and antisense oligonucleotides were designed for insertion into the Bbs I site in the pX330 bicistronic expression vector expressing Cas9 and synthetic single-guide RNA (Addgene, Cambridge, MA) as follows: 5′-CACCGGGCGGCCCGACCCCCCAGG-3′ and 5′-AAACCCTGGGGGGTCGGGCCGCCC-3′ for CHMP4B, and 5′-CACCGCAGGCCCAGTACTGCCGCG-3′ and 5′-AAACCGCGGCAGTACTGGGCCTGC-3′ for ALIX to produce pX330-CHMP4B and pX330-ALIX, respectively.

Article Title: Human babesiosis: Indication of a molecular mimicry between thrombospondin domains from a novel Babesia microti BmP53 protein and host platelets molecules
Article Snippet: For cloning, two pairs of oligonucleotide primers with Bam HI and Xho I restriction enzyme sites were designed and used for the amplification of both truncates, BmP53tr1-TSP1 (forward primer: 5′-CGGGATCCGAGAAGATAAAGGATGAA-3′ ; reverse primer: 5′-CGCTCGAGTTATTTATAGCTAGAGCTCAG -3′ ) and BmP53tr2 (forward primer: 5′-CGGGATCCAGGTTAATGAAGCTGCCA-3′ ; reverse primer: 5′-CGCTCGAGTTACGCAAATTGGGCAAATGA-3′ ). .. The PCR products were ligated to the Bam HI and Xho I restriction sites of the glutathione S-transferase (GST) fusion expression vector, pGEX-4T-1 (GE Healthcare Life Sciences, UK). .. The constructs of the resulting plasmids were checked for accurate insertion by digestion with Bam HI and Xho I according to the manufacturer’s instructions (ROCHE Diagnostics, Germany).

Positron Emission Tomography:

Article Title: Structural basis for recruiting and shuttling of the spliceosomal deubiquitinase USP4 by SART3
Article Snippet: Final protein complex was concentrated to 20 mg ml−1 in same buffer using Vivaspin concentrator (Vivagen) for crystallization trials. .. The glutathione S-transferase (GST)-tagged SART3 NLS, comprising residues 601–649, was overexpressed in E. coli strain Rossetta 2(DE3) at 18°C from pGEX-4T-1 (GE Healthcare), and hexa-histidine tagged mouse importin-α2 (SwissProt entry P52293) comprising residues 70–498 (ImpA) was overexpressed separately from the same strain at 18°C from pET-28a. .. After harvesting, the two sets of cells were mixed, suspended in 50 mM Tris-HCl pH 8.0, 150 mM NaCl and 0.1 mM PMSF, and disrupted by sonication on the ice.

Affinity Column:

Article Title: Structural basis for recruiting and shuttling of the spliceosomal deubiquitinase USP4 by SART3
Article Snippet: The glutathione S-transferase (GST)-tagged SART3 NLS, comprising residues 601–649, was overexpressed in E. coli strain Rossetta 2(DE3) at 18°C from pGEX-4T-1 (GE Healthcare), and hexa-histidine tagged mouse importin-α2 (SwissProt entry P52293) comprising residues 70–498 (ImpA) was overexpressed separately from the same strain at 18°C from pET-28a. .. After harvesting, the two sets of cells were mixed, suspended in 50 mM Tris-HCl pH 8.0, 150 mM NaCl and 0.1 mM PMSF, and disrupted by sonication on the ice.

Recombinant:

Article Title: Recombinant Paracoccin Reproduces the Biological Properties of the Native Protein and Induces Protective Th1 Immunity against Paracoccidioides brasiliensis Infection
Article Snippet: First, the largest exon was amplified from P. brasiliensis genomic DNA using the oligonucleotide primers FPADG ( 5′-CTGGATCCATGCAAGCACCCGACCAAC-3′ ) and RPADG ( 5′-CGGGAATTCCTACCAACTCGTTATTGATAGAGCGATAA-3′ ), and then cloned into pGEX-4T-1 (GE Healthcare, Upsala, Sweden) flanked by Bam HI and Eco RI restriction sites. .. In the first strategy, the paracoccin fragment, referred to as rPCNexon4 , is fused to a GST tag in the pGEX vector, whereas in the second strategy, the 5′-UTR region, which drives transcription of paracoccin (rPCNfull ) was also synthesized.

Article Title: High yield production of pigeon circovirus capsid protein in the E. coli by evaluating the key parameters needed for protein expression
Article Snippet: Paragraph title: Construction of the recombinant plasmids ... This cDNA was cloned into either pET28a, pET32a (Novagen, Madison, WI) or pGEX-4T-1 (GE Healthcare, Piscataway, NJ) individually using Eco R1 and Xho I (Takara, Japan) restriction sites.

Article Title: Plasmodium falciparum ookinete expression of plasmepsin VII and plasmepsin X
Article Snippet: Paragraph title: Production of recombinant PfPM VII and PfPM X in Escherichia coli ... PfPM VII and PfPM X genes were then cloned into expression vectors pET32 and pGEX 4T-1 (GE Healthcare, Piscataway, NJ, USA), respectively.

Article Title: Precision Targeting of BFL-1/A1 and an ATM Co-dependency in Human Cancer
Article Snippet: Protein purity and identity was confirmed by Coomassie staining and western blot analysis using a mouse monoclonal anti-His6 tag antibody (Abcam). .. Recombinant anti-apoptotic BCL-XL ΔC (aa 1–212) and MCL-1ΔNΔC (aa 172–329) were cloned into the pGEX-4T-1 (GE Healthcare, N-terminal GST tag) expression vector, expressed in BL21(DE3) Escherichia coli (Sigma Aldrich), and purified as described previously ( ). .. Protein expression was induced using 0.5 mM IPTG overnight at 37°C.

Article Title: Plasmodium berghei Circumvents Immune Responses Induced by Merozoite Surface Protein 1- and Apical Membrane Antigen 1-Based Vaccines
Article Snippet: Paragraph title: Recombinant proteins ... Each of these DNA fragments was cloned into the EcoR I/Sma I sites of pGEX-4T-1 (GE Healthcare UK Limited, Buckinghamshire, UK).

Gel Extraction:

Article Title: Plasmodium falciparum ookinete expression of plasmepsin VII and plasmepsin X
Article Snippet: PCR products were gel purified on a 0.8 % agarose gel using the PureLink gel extraction kit (Invitrogen, Carlsbad, CA, USA), ligated into pCR4-TOPO (Invitrogen, Carlsbad, CA, USA), transformed into Top10 competent cells (Invitrogen, Carlsbad, CA, USA) and sequence-verified (Eton Bioscience, San Diego, CA, USA). .. PfPM VII and PfPM X genes were then cloned into expression vectors pET32 and pGEX 4T-1 (GE Healthcare, Piscataway, NJ, USA), respectively.

Activated Clotting Time Assay:

Article Title: Does Variation of the Inter-Domain Linker Sequence Modulate the Metal Binding Behaviour of Helix pomatia Cd-Metallothionein?
Article Snippet: Two HpCdMT mutants were designed to replace the only two residues (-KT-) that act as a linker between the two nine-Cys domains in the HpCdMT sequence with the corresponding linkers from the (giant keyhole limpet) M. crenulata MT [ ] (-VKTEAKTT-; HpCdMcMT protein) or from wheat (T. aestivum ) Ec-1 MT [ ] (-SARSGAAA-; HpCdPlMT protein) ( ). .. They were subsequently digested with BamH I and Xho I restriction enzymes, and the resulting products were ligated in-frame (DNA ligation kit, Takara Bio, Kusatsu, Shiga, Japan) in the pGEX-4T-1 (Amersham-GE Healthcare Europe, Cerdanyola del Valles, Spain) E. coli expression vector, which yields GST-fusion proteins.

Mouse Assay:

Article Title: Plasmodium berghei Circumvents Immune Responses Induced by Merozoite Surface Protein 1- and Apical Membrane Antigen 1-Based Vaccines
Article Snippet: Each of these DNA fragments was cloned into the EcoR I/Sma I sites of pGEX-4T-1 (GE Healthcare UK Limited, Buckinghamshire, UK). .. GST-PyMSP119 was used as an immunogen for vaccination and antigen for ELISA as described previously .

SDS Page:

Article Title: C3G forms complexes with Bcr-Abl and p38? MAPK at the focal adhesions in chronic myeloid leukemia cells: implication in the regulation of leukemic cell adhesion
Article Snippet: To do so, fragments were amplified by PCR and cloned into Eco RI-Xho I sites of pGEX-4T-1 (GE Healthcare Life Sciences). .. Pull-downs were carried out by incubating 1 mg of protein extract in lysis buffer with 12 –μg of GST-fusion proteins, bound to glutathione-sepharose 4 fast flow beads (GE Healthcare Life Sciences), for 2 hours at 4°C.

Plasmid Preparation:

Article Title: Recombinant Paracoccin Reproduces the Biological Properties of the Native Protein and Induces Protective Th1 Immunity against Paracoccidioides brasiliensis Infection
Article Snippet: First, the largest exon was amplified from P. brasiliensis genomic DNA using the oligonucleotide primers FPADG ( 5′-CTGGATCCATGCAAGCACCCGACCAAC-3′ ) and RPADG ( 5′-CGGGAATTCCTACCAACTCGTTATTGATAGAGCGATAA-3′ ), and then cloned into pGEX-4T-1 (GE Healthcare, Upsala, Sweden) flanked by Bam HI and Eco RI restriction sites. .. Second, the full-length, predicted ORF encoding paracoccin was synthesized, and cloned into the pUC57 vector (GenScript, Piscataway, NJ, USA).

Article Title: F-Box Protein FBXO31 Mediates Cyclin D1 Degradation to Induce G1 Arrest Following DNA Damage
Article Snippet: The plasmid pLNCX2-FBXO31 was used as a template to generate FBXO31 mutants using the QuikChange XL site-directed mutagenesis kit (Stratagene). .. To generate in-frame GST-tagged fusion proteins, the wild type and mutant FBXO31 sequences were PCR amplified (using primers WT1_forward 5’-CGGGATCCGCTGCCACAGTGGAGTGC-3’ and WT1_reverse 5’-GGAATTCGATGAGGTCGTCGGGGCG-3’, or WT2_forward 5’-CGGAATTCCGG ATCCAGCTGCCCGAC-3’ and WT2_reverse 5’-ACGTCGACCACGGGCAGCACG AACGG-3’), digested with BamH1 and EcoR1, and cloned into pGEX-4T-1 (Amersham).

Article Title: Bioengineered bacterial vesicles as biological nano-heaters for optoacoustic imaging
Article Snippet: We anticipate that the OMVs presented in this work will be a starting point for future theranostic studies focused on simultaneous optoacoustic imaging and cancer therapeutics. .. Plasmid construction, bacterial strain and growth The melA gene of Rhizobium etli encoding tyrosinase (kindly provided by Professor Guillermo Gosset, Universidad Nacional Autónoma de México, Mexico) was cloned into pGEX-4T-1 (GE Healthcare, Freiburg, Germany). .. The construct pGEX-4T-1-melA was transformed into msbB mutant W3110-K12 E. coli (kindly provided by Professor Sangyong Jon, KAIST, South Korea) .

Article Title: Analysis of Metal-Binding Features of the Wild Type and Two Domain-Truncated Mutant Variants of Littorina littorea Metallothionein Reveals Its Cd-Specific Character
Article Snippet: The LlwtMT, Lltr1MT, and Lltr2MT coding sequences designed this way were purchased as synthetic DNAs IDT & Conda Labs (Madrid, Spain). .. After PCR amplification (35 cycles: 95 °C 30 s, 50 °C 30 s, and 72 °C 30 s, using Expand High Fidelity (Roche Diagnostics S.L:, San Cugat del Vallès, Spain) thermostable DNA polymerase) of the synthetic cDNAs using the flanking primers 5′-TTTATTGGATCCATGAGCTC-3′ (LlwtMT forward); 5′-TTTCTCGAGTCATTTGCATG-3′ (LlwtMT reverse); 5′-TTTATTGGATCCGGTAAGGG-3′ (Lltr1MT forward); 5′-TTTCTCGAGTTACTTACAGG-3′ (Lltr1MT reverse); 5′-TTTATTGGATCCGGCAAAGGG-3′ (Lltr2MT forward); and 5′-TTTCTCGAGTTATTTGCAAG-3′ (Lltr2MT reverse), they were subsequently digested with BamHI and XhoI restriction enzymes, and the resulting products were ligated in-frame (DNA ligation kit, Takara Bio, Kusatsu, Shiga, Japan) in the pGEX-4T-1 (Amersham-GE Healthcare Europe, Cerdanyola del Valles, Spain) E. coli expression vector, which yields GST-fusion proteins. .. DNA sequencing allowed confirming of all the DNA constructs (ABIPRISM 310, Applied Biosystems, Foster City, CA, USA) using the BigDye Terminator.

Article Title: Does Variation of the Inter-Domain Linker Sequence Modulate the Metal Binding Behaviour of Helix pomatia Cd-Metallothionein?
Article Snippet: After PCR amplification (35 cycles: 95 °C 30 s, 50 °C 30 s, and 72 °C 30 s, using Expand High Fidelity (Roche) thermostable DNA polymerase) of the synthetic cDNAs using the flanking primers: 5’-TTTATTGGATCCGGTAAGGG-3’ (HpCdMcMT forward); 5’-TTTCTCGAGTTACTTACAGG-3’ (HpCdMcMT reverse); 5’-TTTATTGGATCCGGCAAAGGG-3’ (HpCdPlMT forward); and 5’-TTTCTCGAGTTATTTGCAAG-3’ (HpCdPlMT reverse). .. They were subsequently digested with BamH I and Xho I restriction enzymes, and the resulting products were ligated in-frame (DNA ligation kit, Takara Bio, Kusatsu, Shiga, Japan) in the pGEX-4T-1 (Amersham-GE Healthcare Europe, Cerdanyola del Valles, Spain) E. coli expression vector, which yields GST-fusion proteins. .. DNA sequencing allow confirming all the DNA constructs (ABIPRISM 310, Applied Biosystems, Foster City, CA, USA), using BigDye Terminator.

Article Title: Plasmodium falciparum ookinete expression of plasmepsin VII and plasmepsin X
Article Snippet: PfPM VII and PfPM X genes were then cloned into expression vectors pET32 and pGEX 4T-1 (GE Healthcare, Piscataway, NJ, USA), respectively. .. The recombinant PfPM VII-HIS-tagged (rPfPM VII-HIS) and PfPM X-GST-tagged (rPfPM X-GST) fusion proteins were expressed in Rosetta (Merck/Novagen, Darmstadt, Germany) competent cells according to standard protocol [ ].

Article Title: Nuclear GSK3β promotes tumorigenesis by phosphorylating KDM1A and inducing its deubiquitination by USP22
Article Snippet: Paragraph title: Plasmid constructs and mutagenesis ... GST-tagged KDM1A or KDM1A S683A/S687A was constructed by cloning the wild-type or mutant cDNA into pGEX-4T-1 (GE Healthcare).

Article Title: ESCRT-III mediates budding across the inner nuclear membrane and regulates its integrity
Article Snippet: To generate fusion proteins of GST with the ALIX Bro domain (amino acids 1–358) and the ALIX V domain (amino acids 362–702) (GST-ALIX-Bro and GST-ALIX-V, respectively), pGEX-ALIX-Bro and pGEX-ALIX-V were constructed by cloning the appropriate ALI X domain, amplified by PCR from pCI-FLAG-ALIX, and cloned into pGEX-4T-1 (GE Healthcare Bio-Sciences, Piscataway, NJ). .. Sense and antisense oligonucleotides were designed for insertion into the Bbs I site in the pX330 bicistronic expression vector expressing Cas9 and synthetic single-guide RNA (Addgene, Cambridge, MA) as follows: 5′-CACCGGGCGGCCCGACCCCCCAGG-3′ and 5′-AAACCCTGGGGGGTCGGGCCGCCC-3′ for CHMP4B, and 5′-CACCGCAGGCCCAGTACTGCCGCG-3′ and 5′-AAACCGCGGCAGTACTGGGCCTGC-3′ for ALIX to produce pX330-CHMP4B and pX330-ALIX, respectively.

Article Title: Human babesiosis: Indication of a molecular mimicry between thrombospondin domains from a novel Babesia microti BmP53 protein and host platelets molecules
Article Snippet: For cloning, two pairs of oligonucleotide primers with Bam HI and Xho I restriction enzyme sites were designed and used for the amplification of both truncates, BmP53tr1-TSP1 (forward primer: 5′-CGGGATCCGAGAAGATAAAGGATGAA-3′ ; reverse primer: 5′-CGCTCGAGTTATTTATAGCTAGAGCTCAG -3′ ) and BmP53tr2 (forward primer: 5′-CGGGATCCAGGTTAATGAAGCTGCCA-3′ ; reverse primer: 5′-CGCTCGAGTTACGCAAATTGGGCAAATGA-3′ ). .. The PCR products were ligated to the Bam HI and Xho I restriction sites of the glutathione S-transferase (GST) fusion expression vector, pGEX-4T-1 (GE Healthcare Life Sciences, UK). .. The constructs of the resulting plasmids were checked for accurate insertion by digestion with Bam HI and Xho I according to the manufacturer’s instructions (ROCHE Diagnostics, Germany).

Article Title: Transcriptional regulation of autophagy-lysosomal function in BRAF-driven melanoma progression and chemoresistance
Article Snippet: Flag-tagged BRAFV600E was cloned into pCDH-CMV-MCS-EF1-puro backbone (System Biosciences; SBI) to generate lentiviral transfer constructs. shRNA-resistant TFEB mutants were generated using site-directed mutagenesis (Q5 0552S, New England Biolab). .. For expression of GST fusion in Escherichia coli BL21 cells, the full-length TFEB, pro-TGFβ, and its point mutants were cloned into the pGEX-4T-1 (Pharmacia Amersham) vector or the pET32a (EMD Biosciences) vector. .. The plasmids of ZKSCAN3-V5 was provided by Dr. Pinghui Feng, University of Southern California, USA.

Article Title: Precision Targeting of BFL-1/A1 and an ATM Co-dependency in Human Cancer
Article Snippet: Protein purity and identity was confirmed by Coomassie staining and western blot analysis using a mouse monoclonal anti-His6 tag antibody (Abcam). .. Recombinant anti-apoptotic BCL-XL ΔC (aa 1–212) and MCL-1ΔNΔC (aa 172–329) were cloned into the pGEX-4T-1 (GE Healthcare, N-terminal GST tag) expression vector, expressed in BL21(DE3) Escherichia coli (Sigma Aldrich), and purified as described previously ( ). .. Protein expression was induced using 0.5 mM IPTG overnight at 37°C.

shRNA:

Article Title: Transcriptional regulation of autophagy-lysosomal function in BRAF-driven melanoma progression and chemoresistance
Article Snippet: Flag-tagged BRAFV600E was cloned into pCDH-CMV-MCS-EF1-puro backbone (System Biosciences; SBI) to generate lentiviral transfer constructs. shRNA-resistant TFEB mutants were generated using site-directed mutagenesis (Q5 0552S, New England Biolab). .. For expression of GST fusion in Escherichia coli BL21 cells, the full-length TFEB, pro-TGFβ, and its point mutants were cloned into the pGEX-4T-1 (Pharmacia Amersham) vector or the pET32a (EMD Biosciences) vector.

Agarose Gel Electrophoresis:

Article Title: Plasmodium falciparum ookinete expression of plasmepsin VII and plasmepsin X
Article Snippet: PCR products were gel purified on a 0.8 % agarose gel using the PureLink gel extraction kit (Invitrogen, Carlsbad, CA, USA), ligated into pCR4-TOPO (Invitrogen, Carlsbad, CA, USA), transformed into Top10 competent cells (Invitrogen, Carlsbad, CA, USA) and sequence-verified (Eton Bioscience, San Diego, CA, USA). .. PfPM VII and PfPM X genes were then cloned into expression vectors pET32 and pGEX 4T-1 (GE Healthcare, Piscataway, NJ, USA), respectively.

In Vitro:

Article Title: CIB1 is a Regulator of Pathological Cardiac Hypertrophy
Article Snippet: To generate GST fusion proteins, CIB1 cDNA sequences were amplified by PCR and cloned into pGEX-4T-1 (Amersham Pharmacia Biotech). .. To generate GST fusion proteins, CIB1 cDNA sequences were amplified by PCR and cloned into pGEX-4T-1 (Amersham Pharmacia Biotech).

Article Title: Polarized Exocytosis Induces Compensatory Endocytosis by Sec4p-Regulated Cortical Actin Polymerization
Article Snippet: Paragraph title: In Vitro Pyrene-Actin Polymerization Assay and Affinity Purification of Fusion Proteins ... For purification of GST-fusion proteins from bacterial extracts, and their subsequent use in actin polymerization assays, coding sequences for SLA1 , LAS17 , SEC4 , and YPT1 were sub-cloned into Bam HI and Sal I sites in pGEX-4T-1 (GE Healthcare Life Sciences, Pittsburg, PA).

Selection:

Article Title: Human babesiosis: Indication of a molecular mimicry between thrombospondin domains from a novel Babesia microti BmP53 protein and host platelets molecules
Article Snippet: Paragraph title: Selection and cloning of BmP53 gene truncates into pGEX-4T-1 vector ... The PCR products were ligated to the Bam HI and Xho I restriction sites of the glutathione S-transferase (GST) fusion expression vector, pGEX-4T-1 (GE Healthcare Life Sciences, UK).

Affinity Chromatography:

Article Title: Precision Targeting of BFL-1/A1 and an ATM Co-dependency in Human Cancer
Article Snippet: Recombinant full-length BFL-1 (aa 1-175) containing an N-terminal hexahistidine tag was cloned into the PTYB1 vector (New England Biolabs) and purified as above except for the addition of a chitin affinity chromatography and DTT elution step. .. Recombinant anti-apoptotic BCL-XL ΔC (aa 1–212) and MCL-1ΔNΔC (aa 172–329) were cloned into the pGEX-4T-1 (GE Healthcare, N-terminal GST tag) expression vector, expressed in BL21(DE3) Escherichia coli (Sigma Aldrich), and purified as described previously ( ).

Produced:

Article Title: Bioengineered bacterial vesicles as biological nano-heaters for optoacoustic imaging
Article Snippet: Plasmid construction, bacterial strain and growth The melA gene of Rhizobium etli encoding tyrosinase (kindly provided by Professor Guillermo Gosset, Universidad Nacional Autónoma de México, Mexico) was cloned into pGEX-4T-1 (GE Healthcare, Freiburg, Germany). .. Plasmid construction, bacterial strain and growth The melA gene of Rhizobium etli encoding tyrosinase (kindly provided by Professor Guillermo Gosset, Universidad Nacional Autónoma de México, Mexico) was cloned into pGEX-4T-1 (GE Healthcare, Freiburg, Germany).

Article Title: Plasmodium berghei Circumvents Immune Responses Induced by Merozoite Surface Protein 1- and Apical Membrane Antigen 1-Based Vaccines
Article Snippet: Each of these DNA fragments was cloned into the EcoR I/Sma I sites of pGEX-4T-1 (GE Healthcare UK Limited, Buckinghamshire, UK). .. These recombinant proteins were recognized by P. yoelii or P. berghei -hyperimmune sera, which were obtained from BALB/c mice that had recovered from repeated infections of the corresponding parasite following treatment with chloroquine as described previously .

FLAG-tag:

Article Title: ESCRT-III mediates budding across the inner nuclear membrane and regulates its integrity
Article Snippet: ALIX cDNA was cloned into pcDNA3.1( + ) (Invitrogen, Carlsbad, CA) in frame with a Flag-epitope to generate pcDNA3.1-Flag-ALIX. pcDNA3.1-Flag-ALIX-I212D was constructed by replacing ALIX codons I212 in pcDNA3.1-Flag-ALIX with aspartic acid codons as reported previously . .. To generate fusion proteins of GST with the ALIX Bro domain (amino acids 1–358) and the ALIX V domain (amino acids 362–702) (GST-ALIX-Bro and GST-ALIX-V, respectively), pGEX-ALIX-Bro and pGEX-ALIX-V were constructed by cloning the appropriate ALI X domain, amplified by PCR from pCI-FLAG-ALIX, and cloned into pGEX-4T-1 (GE Healthcare Bio-Sciences, Piscataway, NJ).

Lysis:

Article Title: C3G forms complexes with Bcr-Abl and p38? MAPK at the focal adhesions in chronic myeloid leukemia cells: implication in the regulation of leukemic cell adhesion
Article Snippet: To do so, fragments were amplified by PCR and cloned into Eco RI-Xho I sites of pGEX-4T-1 (GE Healthcare Life Sciences). .. GST-Abl-SH3 and GST-C3G-SH3-b constructs have been described previously [ ].

Article Title: Full length RTN3 regulates turnover of tubular endoplasmic reticulum via selective autophagy
Article Snippet: LC3s, GABARAPs and Ub proteins were cloned, as GST fusion proteins, into pGEX-4T-1 (GE Healthcare; Little Chalfont, UK) and expressed in Escherichia coli BL21 (DE3) cells in LB medium. .. Expression was induced by addition of 0.5 mM IPTG and cells were incubated at 37°C for 5 hr.

Article Title: Precision Targeting of BFL-1/A1 and an ATM Co-dependency in Human Cancer
Article Snippet: The column was then sequentially washed with lysis buffer containing 10 mM, 20 mM, 35 mM, and 50 mM imidazole. .. Recombinant anti-apoptotic BCL-XL ΔC (aa 1–212) and MCL-1ΔNΔC (aa 172–329) were cloned into the pGEX-4T-1 (GE Healthcare, N-terminal GST tag) expression vector, expressed in BL21(DE3) Escherichia coli (Sigma Aldrich), and purified as described previously ( ).

Staining:

Article Title: Precision Targeting of BFL-1/A1 and an ATM Co-dependency in Human Cancer
Article Snippet: Protein purity and identity was confirmed by Coomassie staining and western blot analysis using a mouse monoclonal anti-His6 tag antibody (Abcam). .. Recombinant anti-apoptotic BCL-XL ΔC (aa 1–212) and MCL-1ΔNΔC (aa 172–329) were cloned into the pGEX-4T-1 (GE Healthcare, N-terminal GST tag) expression vector, expressed in BL21(DE3) Escherichia coli (Sigma Aldrich), and purified as described previously ( ).

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  • 97
    GE Healthcare plasmid pgex 4t 1
    VP1 protein expression in E. coli using various expression vectors . The VP1 protein expression in E. coli BL21(DE3) using the expression vectors, pET28a (A) and <t>pGEX-4T-1</t> (B) was analyzed by SDS-PAGE and Western-blotting. Anti-His tag and anti-GST tag monoclonal antibody were respectively used for the recognition of differently tagged VP1 proteins expressed by the different expression vectors used. Lane M, pre-stained protein marker; lane 1 and lane 3, before IPTG induction; lane 2 and 4, after IPTG induction for 4 h cultivation. The solid triangle pinpoints the expressed VP1 protein.
    Plasmid Pgex 4t 1, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 97/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/plasmid pgex 4t 1/product/GE Healthcare
    Average 97 stars, based on 33 article reviews
    Price from $9.99 to $1999.99
    plasmid pgex 4t 1 - by Bioz Stars, 2019-10
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    99
    GE Healthcare gst fusion proteins
    ALS-FTLD-associated SQSTM1 mutations impact on the recognition of LC3B (SQSTM1 L341V ) or ubiquitin (SQSTM1 <t>G425R</t> ) in vitro. Mutations as indicated (or wild type, WT) were introduced into the full-length <t>GST-SQSTM1</t> sequence and affinity isolation assays (LC3B and ubiquitin on beads) were performed at 37°C. Bacterial lysates containing the GST-SQSTM1 fusions were incubated with glutathione- (G), control- (C), LC3B (LC3), and ubiquitin-Sepharose (Ub) beads and captured proteins were detected by western blotting (anti-SQSTM1 antibodies). A representative blot is shown; see Fig. S1 for quantification of 3 independent experiments.
    Gst Fusion Proteins, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gst fusion proteins/product/GE Healthcare
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    Price from $9.99 to $1999.99
    gst fusion proteins - by Bioz Stars, 2019-10
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    VP1 protein expression in E. coli using various expression vectors . The VP1 protein expression in E. coli BL21(DE3) using the expression vectors, pET28a (A) and pGEX-4T-1 (B) was analyzed by SDS-PAGE and Western-blotting. Anti-His tag and anti-GST tag monoclonal antibody were respectively used for the recognition of differently tagged VP1 proteins expressed by the different expression vectors used. Lane M, pre-stained protein marker; lane 1 and lane 3, before IPTG induction; lane 2 and 4, after IPTG induction for 4 h cultivation. The solid triangle pinpoints the expressed VP1 protein.

    Journal: Microbial Cell Factories

    Article Title: High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development

    doi: 10.1186/1475-2859-10-56

    Figure Lengend Snippet: VP1 protein expression in E. coli using various expression vectors . The VP1 protein expression in E. coli BL21(DE3) using the expression vectors, pET28a (A) and pGEX-4T-1 (B) was analyzed by SDS-PAGE and Western-blotting. Anti-His tag and anti-GST tag monoclonal antibody were respectively used for the recognition of differently tagged VP1 proteins expressed by the different expression vectors used. Lane M, pre-stained protein marker; lane 1 and lane 3, before IPTG induction; lane 2 and 4, after IPTG induction for 4 h cultivation. The solid triangle pinpoints the expressed VP1 protein.

    Article Snippet: Using a specifically designed primer set, forward primer CAV VP1 F: 5'-GCGGAATTCATGGCAAGACGAGCTCGCAGA-3' and reverse primer CAV VP1-R: 5'-GGGCTCGAGTCAGGGCTGCGTCCCCCAGTA-3', respectively containing the EcoR I and Xho I sites, the full-length VP1 gene was amplified using the plasmid pET28a-VP1 as template DNA, and then cloned into the plasmid pGEX-4T-1 (GE healthcare, Piscataway, NJ), which was then designated to be pGEX-4T-1-VP1 (Figure , panel b).

    Techniques: Expressing, SDS Page, Western Blot, Staining, Marker

    Expression of recombinant GST-VP1 protein in three different E. coli strains . The GST-VP1 protein expression in three E. coli strains, BL21(DE3), BL21-CodonPlus(DE3)-RP and BL21(DE3)-pLysS all containing pGEX-4T-1-VP1 was examined. The GST-VP1 protein was examined and detected using SDS-PAGE (A) and Western-blotting (B). Anti-GST tag monoclonal antibody was used to recognize the VP1 protein. Lane M, pre-stained protein marker; lane 1, 4 and 7, before IPTG induction; lane 2, 5 and 8, after IPTG induction for 1 hrs cultivation; lane 3, 6 and 9, after IPTG induction for 4 h cultivation. The solid triangle pinpoints the expressed VP1 protein.

    Journal: Microbial Cell Factories

    Article Title: High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development

    doi: 10.1186/1475-2859-10-56

    Figure Lengend Snippet: Expression of recombinant GST-VP1 protein in three different E. coli strains . The GST-VP1 protein expression in three E. coli strains, BL21(DE3), BL21-CodonPlus(DE3)-RP and BL21(DE3)-pLysS all containing pGEX-4T-1-VP1 was examined. The GST-VP1 protein was examined and detected using SDS-PAGE (A) and Western-blotting (B). Anti-GST tag monoclonal antibody was used to recognize the VP1 protein. Lane M, pre-stained protein marker; lane 1, 4 and 7, before IPTG induction; lane 2, 5 and 8, after IPTG induction for 1 hrs cultivation; lane 3, 6 and 9, after IPTG induction for 4 h cultivation. The solid triangle pinpoints the expressed VP1 protein.

    Article Snippet: Using a specifically designed primer set, forward primer CAV VP1 F: 5'-GCGGAATTCATGGCAAGACGAGCTCGCAGA-3' and reverse primer CAV VP1-R: 5'-GGGCTCGAGTCAGGGCTGCGTCCCCCAGTA-3', respectively containing the EcoR I and Xho I sites, the full-length VP1 gene was amplified using the plasmid pET28a-VP1 as template DNA, and then cloned into the plasmid pGEX-4T-1 (GE healthcare, Piscataway, NJ), which was then designated to be pGEX-4T-1-VP1 (Figure , panel b).

    Techniques: Expressing, Recombinant, SDS Page, Western Blot, Staining, Marker

    Growth kinetics and production profiles of GST-VP1 protein in three E. coli strains . (A) Growth profile of BL21(DE3), BL21-CodonPlus(DE3)-RP and BL21(DE3)-pLysS in LB medium post-induction. The production profiles of the three E. coli strains, (B)BL21(DE3)-pLysS, (C)BL21(DE3) and (D)BL21-CodonPlus(DE3)-RP containing pGEX-4T-1-VP1 are shown over time course after IPTG induction.

    Journal: Microbial Cell Factories

    Article Title: High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development

    doi: 10.1186/1475-2859-10-56

    Figure Lengend Snippet: Growth kinetics and production profiles of GST-VP1 protein in three E. coli strains . (A) Growth profile of BL21(DE3), BL21-CodonPlus(DE3)-RP and BL21(DE3)-pLysS in LB medium post-induction. The production profiles of the three E. coli strains, (B)BL21(DE3)-pLysS, (C)BL21(DE3) and (D)BL21-CodonPlus(DE3)-RP containing pGEX-4T-1-VP1 are shown over time course after IPTG induction.

    Article Snippet: Using a specifically designed primer set, forward primer CAV VP1 F: 5'-GCGGAATTCATGGCAAGACGAGCTCGCAGA-3' and reverse primer CAV VP1-R: 5'-GGGCTCGAGTCAGGGCTGCGTCCCCCAGTA-3', respectively containing the EcoR I and Xho I sites, the full-length VP1 gene was amplified using the plasmid pET28a-VP1 as template DNA, and then cloned into the plasmid pGEX-4T-1 (GE healthcare, Piscataway, NJ), which was then designated to be pGEX-4T-1-VP1 (Figure , panel b).

    Techniques:

    Growth kinetics and production profiles of GST-opt-VP1 protein in E. coli BL21(DE3)-pLysS and BL21(DE3) . (A) Growth profile of BL21(DE3) and BL21(DE3)-pLysS in LB medium during post-induction. (B) The production profiles of the E. coli strains, (B)BL21(DE3)-pLysS, and BL21(DE3) containing pGEX-4T-1-Opt-VP1 are shown after induction.

    Journal: Microbial Cell Factories

    Article Title: High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development

    doi: 10.1186/1475-2859-10-56

    Figure Lengend Snippet: Growth kinetics and production profiles of GST-opt-VP1 protein in E. coli BL21(DE3)-pLysS and BL21(DE3) . (A) Growth profile of BL21(DE3) and BL21(DE3)-pLysS in LB medium during post-induction. (B) The production profiles of the E. coli strains, (B)BL21(DE3)-pLysS, and BL21(DE3) containing pGEX-4T-1-Opt-VP1 are shown after induction.

    Article Snippet: Using a specifically designed primer set, forward primer CAV VP1 F: 5'-GCGGAATTCATGGCAAGACGAGCTCGCAGA-3' and reverse primer CAV VP1-R: 5'-GGGCTCGAGTCAGGGCTGCGTCCCCCAGTA-3', respectively containing the EcoR I and Xho I sites, the full-length VP1 gene was amplified using the plasmid pET28a-VP1 as template DNA, and then cloned into the plasmid pGEX-4T-1 (GE healthcare, Piscataway, NJ), which was then designated to be pGEX-4T-1-VP1 (Figure , panel b).

    Techniques:

    Schematic diagram of the constructs used for CVA VP1 protein expression . (A) Schematic representation of the VP1 protein variants and expression vectors used in this study. The designations of the VP1 protein and its expression vectors are indicated, a, b and c. The first two constructs, a and b, contained the full-length VP1 gene cloned into vectors pET28a and pGEX-4T-1, for expression of VP1 protein with a six-histidine (6 × His) tag and glutathione-s-transferase (GST) tag at its N-terminus, respectively. Construct c contained VP1 gene with codon-optimized at its N-terminus; this was derived from construct b by replacing the rare codons in the area 1-321 bp without altering amino acid sequence. The N-terminal codon-optimized VP1 gene, opt-N, was fused with C-terminal domain of VP1 gene (VP1 C-term) by overlapping PCR, and then cloned into pGEX-4T-1. (B) Sequence comparison between the WT VP1 and Opt VP1 gene. The nucleotide sequences were compared between the original VP1 gene (WT VP1) and the sequence of codon-optimized VP1 gene (Opt VP1) over the N-terminal region. An asterisk ( * ) represents the fact that the aligned nucleotides are identical.

    Journal: Microbial Cell Factories

    Article Title: High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development

    doi: 10.1186/1475-2859-10-56

    Figure Lengend Snippet: Schematic diagram of the constructs used for CVA VP1 protein expression . (A) Schematic representation of the VP1 protein variants and expression vectors used in this study. The designations of the VP1 protein and its expression vectors are indicated, a, b and c. The first two constructs, a and b, contained the full-length VP1 gene cloned into vectors pET28a and pGEX-4T-1, for expression of VP1 protein with a six-histidine (6 × His) tag and glutathione-s-transferase (GST) tag at its N-terminus, respectively. Construct c contained VP1 gene with codon-optimized at its N-terminus; this was derived from construct b by replacing the rare codons in the area 1-321 bp without altering amino acid sequence. The N-terminal codon-optimized VP1 gene, opt-N, was fused with C-terminal domain of VP1 gene (VP1 C-term) by overlapping PCR, and then cloned into pGEX-4T-1. (B) Sequence comparison between the WT VP1 and Opt VP1 gene. The nucleotide sequences were compared between the original VP1 gene (WT VP1) and the sequence of codon-optimized VP1 gene (Opt VP1) over the N-terminal region. An asterisk ( * ) represents the fact that the aligned nucleotides are identical.

    Article Snippet: Using a specifically designed primer set, forward primer CAV VP1 F: 5'-GCGGAATTCATGGCAAGACGAGCTCGCAGA-3' and reverse primer CAV VP1-R: 5'-GGGCTCGAGTCAGGGCTGCGTCCCCCAGTA-3', respectively containing the EcoR I and Xho I sites, the full-length VP1 gene was amplified using the plasmid pET28a-VP1 as template DNA, and then cloned into the plasmid pGEX-4T-1 (GE healthcare, Piscataway, NJ), which was then designated to be pGEX-4T-1-VP1 (Figure , panel b).

    Techniques: Construct, Expressing, Clone Assay, Derivative Assay, Sequencing, Polymerase Chain Reaction

    Electrophoretic and SDS-PAGE analysis of recombinant pGB2B3. (A) Electrophoretic identification of digested pGB2B3 recombinant (1.0% agarose gel). Lane M, DL2000 marker; lane 1, pGB2B3/BamHI+EcoRI; lane 2, pGEX-4T-1 plasmid. (B) Electrophoresis of the PCR product from pGB2B3 (1.0% agarose gel). Lane M, 50 bp marker; lane 1, PCR product of pGB2B3 plasmid; lane 2, PCR product of pGEX-4T-1 plasmid. (C) SDS-PAGE analysis of GST-tagged BD2/3 fusion protein expression. Lane M, protein MW marker; lane 1, expression products of pGEX-4T-1 induced by IPTG; lane 2, expression products of pGB2B3 induced by IPTG; lane 3, supernatant of lysed culture of pGB2B3 induced by IPTG; lane 4, pellet of lysed culture of pGB2B3 induced by IPTG; lane 5, expression products of pGB2B3 without IPTG induction. BD, β-defensin; IPTG, isopropyl-β-D-thiogalactopyranoside; GST, glutathione S transferase.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Improved expression of recombinant fusion defensin gene plasmids packed with chitosan-derived nanoparticles and effect on antibacteria and mouse immunity

    doi: 10.3892/etm.2018.6716

    Figure Lengend Snippet: Electrophoretic and SDS-PAGE analysis of recombinant pGB2B3. (A) Electrophoretic identification of digested pGB2B3 recombinant (1.0% agarose gel). Lane M, DL2000 marker; lane 1, pGB2B3/BamHI+EcoRI; lane 2, pGEX-4T-1 plasmid. (B) Electrophoresis of the PCR product from pGB2B3 (1.0% agarose gel). Lane M, 50 bp marker; lane 1, PCR product of pGB2B3 plasmid; lane 2, PCR product of pGEX-4T-1 plasmid. (C) SDS-PAGE analysis of GST-tagged BD2/3 fusion protein expression. Lane M, protein MW marker; lane 1, expression products of pGEX-4T-1 induced by IPTG; lane 2, expression products of pGB2B3 induced by IPTG; lane 3, supernatant of lysed culture of pGB2B3 induced by IPTG; lane 4, pellet of lysed culture of pGB2B3 induced by IPTG; lane 5, expression products of pGB2B3 without IPTG induction. BD, β-defensin; IPTG, isopropyl-β-D-thiogalactopyranoside; GST, glutathione S transferase.

    Article Snippet: The fusion gene BD2/3 was digested with Bam HI and Eco RI, and then cloned into the pGEX-4T-1 expression plasmid (Pharmacia Biotech; GE Healthcare, Chicago, IL, USA), a prokaryotic expression vector containing the Tac promoter and a glutathione S transferase (GST) tag sequence for fusion protein expression.

    Techniques: SDS Page, Recombinant, Agarose Gel Electrophoresis, Marker, Plasmid Preparation, Electrophoresis, Polymerase Chain Reaction, Expressing

    Electrophoretic and SDS-PAGE analysis of recombinant pGB2B3. (A) Electrophoretic identification of digested pGB2B3 recombinant (1.0% agarose gel). Lane M, DL2000 marker; lane 1, pGB2B3/BamHI+EcoRI; lane 2, pGEX-4T-1 plasmid. (B) Electrophoresis of the PCR product from pGB2B3 (1.0% agarose gel). Lane M, 50 bp marker; lane 1, PCR product of pGB2B3 plasmid; lane 2, PCR product of pGEX-4T-1 plasmid. (C) SDS-PAGE analysis of GST-tagged BD2/3 fusion protein expression. Lane M, protein MW marker; lane 1, expression products of pGEX-4T-1 induced by IPTG; lane 2, expression products of pGB2B3 induced by IPTG; lane 3, supernatant of lysed culture of pGB2B3 induced by IPTG; lane 4, pellet of lysed culture of pGB2B3 induced by IPTG; lane 5, expression products of pGB2B3 without IPTG induction. BD, β-defensin; IPTG, isopropyl-β-D-thiogalactopyranoside; GST, glutathione S transferase.

    Journal: Experimental and Therapeutic Medicine

    Article Title: Improved expression of recombinant fusion defensin gene plasmids packed with chitosan-derived nanoparticles and effect on antibacteria and mouse immunity

    doi: 10.3892/etm.2018.6716

    Figure Lengend Snippet: Electrophoretic and SDS-PAGE analysis of recombinant pGB2B3. (A) Electrophoretic identification of digested pGB2B3 recombinant (1.0% agarose gel). Lane M, DL2000 marker; lane 1, pGB2B3/BamHI+EcoRI; lane 2, pGEX-4T-1 plasmid. (B) Electrophoresis of the PCR product from pGB2B3 (1.0% agarose gel). Lane M, 50 bp marker; lane 1, PCR product of pGB2B3 plasmid; lane 2, PCR product of pGEX-4T-1 plasmid. (C) SDS-PAGE analysis of GST-tagged BD2/3 fusion protein expression. Lane M, protein MW marker; lane 1, expression products of pGEX-4T-1 induced by IPTG; lane 2, expression products of pGB2B3 induced by IPTG; lane 3, supernatant of lysed culture of pGB2B3 induced by IPTG; lane 4, pellet of lysed culture of pGB2B3 induced by IPTG; lane 5, expression products of pGB2B3 without IPTG induction. BD, β-defensin; IPTG, isopropyl-β-D-thiogalactopyranoside; GST, glutathione S transferase.

    Article Snippet: The fusion gene BD2/3 was digested with Bam HI and Eco RI, and then cloned into the pGEX-4T-1 expression plasmid (Pharmacia Biotech; GE Healthcare, Chicago, IL, USA), a prokaryotic expression vector containing the Tac promoter and a glutathione S transferase (GST) tag sequence for fusion protein expression.

    Techniques: SDS Page, Recombinant, Agarose Gel Electrophoresis, Marker, Plasmid Preparation, Electrophoresis, Polymerase Chain Reaction, Expressing

    ALS-FTLD-associated SQSTM1 mutations impact on the recognition of LC3B (SQSTM1 L341V ) or ubiquitin (SQSTM1 G425R ) in vitro. Mutations as indicated (or wild type, WT) were introduced into the full-length GST-SQSTM1 sequence and affinity isolation assays (LC3B and ubiquitin on beads) were performed at 37°C. Bacterial lysates containing the GST-SQSTM1 fusions were incubated with glutathione- (G), control- (C), LC3B (LC3), and ubiquitin-Sepharose (Ub) beads and captured proteins were detected by western blotting (anti-SQSTM1 antibodies). A representative blot is shown; see Fig. S1 for quantification of 3 independent experiments.

    Journal: Autophagy

    Article Title: Defective recognition of LC3B by mutant SQSTM1/p62 implicates impairment of autophagy as a pathogenic mechanism in ALS-FTLD

    doi: 10.1080/15548627.2016.1170257

    Figure Lengend Snippet: ALS-FTLD-associated SQSTM1 mutations impact on the recognition of LC3B (SQSTM1 L341V ) or ubiquitin (SQSTM1 G425R ) in vitro. Mutations as indicated (or wild type, WT) were introduced into the full-length GST-SQSTM1 sequence and affinity isolation assays (LC3B and ubiquitin on beads) were performed at 37°C. Bacterial lysates containing the GST-SQSTM1 fusions were incubated with glutathione- (G), control- (C), LC3B (LC3), and ubiquitin-Sepharose (Ub) beads and captured proteins were detected by western blotting (anti-SQSTM1 antibodies). A representative blot is shown; see Fig. S1 for quantification of 3 independent experiments.

    Article Snippet: The plasmids for expression of full-length wild-type and G425R mutant SQSTM1 protein (residues 1 to 440) as GST fusion proteins (pGEX-4T-1; GE Healthcare, 28-9545-49) in E. coli have been described previously.

    Techniques: In Vitro, Sequencing, Isolation, Incubation, Western Blot