Structured Review

GE Healthcare pgex 4t 1
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 <t>pGEX-4T-1.</t> 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.
Pgex 4t 1, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 288 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/pgex 4t 1/product/GE Healthcare
Average 99 stars, based on 288 article reviews
Price from $9.99 to $1999.99
pgex 4t 1 - by Bioz Stars, 2020-02
99/100 stars

Images

1) 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, Sequencing, Binding Assay

2) Product Images from "Identification of dynein light chain road block-1 as a novel interaction partner with the human reduced folate carrier"

Article Title: Identification of dynein light chain road block-1 as a novel interaction partner with the human reduced folate carrier

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

doi: 10.1152/ajpgi.00154.2009

Pull-down assay of hRFC from human intestinal epithelial Caco-2 cells by GST-DYNLRB1. A : generation and purification of GST-DYNLRB1 fusion protein and GST. Extracts from BL-21 Escherichia coli cells harboring recombinant pGEX-4T-1 ( 1 ), purified GST ( 2 ), pGEX-4T-1-DYNLRB1 ( 3 ), purified GST-DYNLRB1 ( 4 ), and marker ( M ). Protein samples were run on SDS-PAGE and stained with Coomassie blue. B : Caco-2 cells lysate was incubated with glutathione-Sepharose 4B beads adsorbed with GST-DYNLRB1 or GST alone (negative control). Proteins bound to the beads were separated, washed and analyzed by Western Blotting using anti-hRFC antibodies. 1 , Caco-2 cell lysate; 2 , proteins bound to GST-DYNLRB1; 3 , proteins bound to unfused GST.
Figure Legend Snippet: Pull-down assay of hRFC from human intestinal epithelial Caco-2 cells by GST-DYNLRB1. A : generation and purification of GST-DYNLRB1 fusion protein and GST. Extracts from BL-21 Escherichia coli cells harboring recombinant pGEX-4T-1 ( 1 ), purified GST ( 2 ), pGEX-4T-1-DYNLRB1 ( 3 ), purified GST-DYNLRB1 ( 4 ), and marker ( M ). Protein samples were run on SDS-PAGE and stained with Coomassie blue. B : Caco-2 cells lysate was incubated with glutathione-Sepharose 4B beads adsorbed with GST-DYNLRB1 or GST alone (negative control). Proteins bound to the beads were separated, washed and analyzed by Western Blotting using anti-hRFC antibodies. 1 , Caco-2 cell lysate; 2 , proteins bound to GST-DYNLRB1; 3 , proteins bound to unfused GST.

Techniques Used: Pull Down Assay, Purification, Recombinant, Marker, SDS Page, Staining, Incubation, Negative Control, Western Blot

3) 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

4) Product Images from "Improved expression of recombinant fusion defensin gene plasmids packed with chitosan-derived nanoparticles and effect on antibacteria and mouse immunity"

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

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2018.6716

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.
Figure Legend 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.

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

5) 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: The international journal of biochemistry & cell biology

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

6) 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

7) Product Images from "Improved expression of recombinant fusion defensin gene plasmids packed with chitosan-derived nanoparticles and effect on antibacteria and mouse immunity"

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

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2018.6716

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.
Figure Legend 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.

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

8) Product Images from "An N-Acyl Homoserine Lactone Synthase in the Ammonia-Oxidizing Bacterium Nitrosospira multiformis"

Article Title: An N-Acyl Homoserine Lactone Synthase in the Ammonia-Oxidizing Bacterium Nitrosospira multiformis

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.03361-13

Bioassay for AHL activity of N. multiformis NmuI. An extract from the recombinant E. coli strain containing pGEX-nmuI was recognized by the reporter strain A. tumefaciens A136 (blue stain). The blank control, E. coli with pGEX-4T-1, had no effect on the
Figure Legend Snippet: Bioassay for AHL activity of N. multiformis NmuI. An extract from the recombinant E. coli strain containing pGEX-nmuI was recognized by the reporter strain A. tumefaciens A136 (blue stain). The blank control, E. coli with pGEX-4T-1, had no effect on the

Techniques Used: Activity Assay, Recombinant, Staining

Related Articles

Clone Assay:

Article Title: An N-Acyl Homoserine Lactone Synthase in the Ammonia-Oxidizing Bacterium Nitrosospira multiformis
Article Snippet: .. Amplified nmuI was cloned into pGEX-4T-1 (GE) as described by the manufacturer, and the resulting recombinant plasmid was termed pGEX-nmuI. .. We also created the LuxR homolog protein (NmuR) expression plasmid pET-R.

Article Title: Ablation of Cypher, a PDZ-LIM domain Z-line protein, causes a severe form of congenital myopathy
Article Snippet: Plasmid construction Plasmids containing distinct parts of mouse α-actinin 2, including the actin-binding domain, Spectrin repeats, or EF hands , were generated by PCR using Pfu DNA polymerase (Stratagene) followed by cloning into pcDNA3FLAG ( ). .. Mutant PDZ domains were generated via PCR-based mutagenesis followed by subcloning into pGEX-4T-1 (Amersham Pharmacia Biotech) or pEGFP-C1 (CLONTECH).

Article Title: The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA
Article Snippet: .. Expression vectors encoding for IQGAP1 C-terminal fragments fused with GST were generated by PCR and cloned into pGEX-4T-1 (GE Healthcare). .. C-terminal deletion mutants of GFP-IQGAP1-T1050AX2 were constructed as follows: ΔCC, a fragment corresponding to aa 1,563–1,657, was amplified by PCR and inserted into pEGFP-IQGAP1-T1050AX2 between an internal SwaI site (corresponding to aa position 1,347) and a SacII site of the 3′-end multiple cloning site fusing aa 1–1,374 with 1,563–1,657; ΔRGC, a fragment corresponding to aa 1–1,562, was amplified by PCR and cloned into the SalI–SacII sites of pEGFP-C3; ΔCC + RGC and pEGFP-IQGAP1-T1050AX2 were cleaved at SwaI (internal) and SmaI 3′-end multiple cloning sites and were self-ligated.

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: 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: KSHV Reactivation from Latency Requires Pim-1 and Pim-3 Kinases to Inactivate the Latency-Associated Nuclear Antigen LANA
Article Snippet: Plasmids Wild-type and kinase-deficient (KD) versions of human Pim-1, Pim-2 and Pim-3 expression vectors were generated by PCR amplification and cloning into the pcDNA3.1/V5-HisC vector . .. The full length and truncated versions of the N-terminal LANA (aa 1–340 (N), aa 100–340 (N6), aa 24–100 (N11), and aa 75–200 (N17)) were generated by PCR amplification from the pCDNA3-LANA and fused to GST expression cassette by ligation to pGEX-4T-1 (GE Healthcare).

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: 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). ..

Amplification:

Article Title: An N-Acyl Homoserine Lactone Synthase in the Ammonia-Oxidizing Bacterium Nitrosospira multiformis
Article Snippet: .. Amplified nmuI was cloned into pGEX-4T-1 (GE) as described by the manufacturer, and the resulting recombinant plasmid was termed pGEX-nmuI. .. We also created the LuxR homolog protein (NmuR) expression plasmid pET-R.

Article Title: The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA
Article Snippet: Expression vectors encoding for IQGAP1 C-terminal fragments fused with GST were generated by PCR and cloned into pGEX-4T-1 (GE Healthcare). .. C-terminal deletion mutants of GFP-IQGAP1-T1050AX2 were constructed as follows: ΔCC, a fragment corresponding to aa 1,563–1,657, was amplified by PCR and inserted into pEGFP-IQGAP1-T1050AX2 between an internal SwaI site (corresponding to aa position 1,347) and a SacII site of the 3′-end multiple cloning site fusing aa 1–1,374 with 1,563–1,657; ΔRGC, a fragment corresponding to aa 1–1,562, was amplified by PCR and cloned into the SalI–SacII sites of pEGFP-C3; ΔCC + RGC and pEGFP-IQGAP1-T1050AX2 were cleaved at SwaI (internal) and SmaI 3′-end multiple cloning sites and were self-ligated.

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: KSHV Reactivation from Latency Requires Pim-1 and Pim-3 Kinases to Inactivate the Latency-Associated Nuclear Antigen LANA
Article Snippet: .. The full length and truncated versions of the N-terminal LANA (aa 1–340 (N), aa 100–340 (N6), aa 24–100 (N11), and aa 75–200 (N17)) were generated by PCR amplification from the pCDNA3-LANA and fused to GST expression cassette by ligation to pGEX-4T-1 (GE Healthcare). .. The luciferase reporter construct in the pGL2-ORF50p backbone containing the RTA promoter (3 kb sequence upstream of the RTA translation initiation codon) was prepared as described before and the luciferase reporter containing seven terminal repeats (TR) of KSHV genome, pGL3-7xTR, was a kind gift from Dr. Rolf Renne (University of Florida) .

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: 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). ..

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: Construction of the recombinant plasmids 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: 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).

Construct:

Article Title: The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA
Article Snippet: GFP-IQGAP1 and -IQGAP1-T1050AX2 expression constructs were gifts from K. Kaibuchi (Nagoya University, Nagoya, Japan). .. Expression vectors encoding for IQGAP1 C-terminal fragments fused with GST were generated by PCR and cloned into pGEX-4T-1 (GE Healthcare).

Article Title: A novel PIP2 binding of ?PKC and its contribution to the neurite induction ability 1
Article Snippet: Paragraph title: Constructs of plasmids encoding GFP-fused εPKC and mutants ... The PCR product was subcloned into pTB701-GFP (described as BS340 in ) for the expression in mammalian cells and into pGEX-4T-1 (Amersham Pharmacia Biotech, Buckinghamshire, UK) for bacterial expression of a glutathione S -transferase (GST)-fusion protein, respectively.

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: 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: KSHV Reactivation from Latency Requires Pim-1 and Pim-3 Kinases to Inactivate the Latency-Associated Nuclear Antigen LANA
Article Snippet: The GST-tagged carboxy-terminal LANA (GST-C-LANA; amino acids (aa) 972-1162) and amino-terminal LANA (GST-N-LANA; aa 1–340 (N-LANA) were expressed from the pGEX4T1-LANA construct. .. The full length and truncated versions of the N-terminal LANA (aa 1–340 (N), aa 100–340 (N6), aa 24–100 (N11), and aa 75–200 (N17)) were generated by PCR amplification from the pCDNA3-LANA and fused to GST expression cassette by ligation to pGEX-4T-1 (GE Healthcare).

Article Title: Overexpression of CERKL, a gene responsible for retinitis pigmentosa in humans, protects cells from apoptosis induced by oxidative stress
Article Snippet: Purification of recombinant proteins To express the four CERKL isoforms as glutathione-S-transferase (GST) fusion proteins, we subcloned each variant in-frame into pGEX-4T-1 (Amersham Biosciences, Chalfont St. Giles, UK), between the XhoI and NotI sites. .. The constructs were transformed into BL21 Codon Plus E.coli cells (Stratagene) to avoid premature protein truncation due to the strongly biased codon usage of the CERKL human gene.

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: Pull-down assays 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).

Incubation:

Article Title: Identification of dynein light chain road block-1 as a novel interaction partner with the human reduced folate carrier
Article Snippet: Following the manufacturer's instructions, GST-DYNLRB1 fusion protein and GST were purified from BL-21 E. coli cells harboring recombinant pGEX-4T-1 and pGEX-4T-1, respectively, by using the Bulk GST Purification Module (Amersham Biosciences, Piscataway, NJ). .. Cleared (14,000 g ) postnuclear extracts (1 mg of protein) were incubated for 3–5 h at 4°C with 50 μg of GST-DYNLRB1 or GST bound to glutathione-Sepharose 4B beads.

Article Title: Arabidopsis FORGETTER1 mediates stress-induced chromatin memory through nucleosome remodeling
Article Snippet: Histone peptide binding assays The PHD domains of FGT1 (aa 667–757) and ING1 ( ) were subcloned into pGEX-4T-1 (Amersham Biosciences, Pittsburgh, PA), expressed in E. coli BL21 (DE3) as GST fusion proteins and purified using Glutathione affinity resins (Thermo Fisher Scientific, Waltham, MA). .. In brief, 1 µg of biotinylated histone peptides (Merck-Millipore, Darmstadt, Germany) were incubated with 10 µg of GST-fusion protein in binding buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 0.1% NP-40, 1 mM PMSF, protease inhibitors) overnight at 4°C with rotation.

Luciferase:

Article Title: KSHV Reactivation from Latency Requires Pim-1 and Pim-3 Kinases to Inactivate the Latency-Associated Nuclear Antigen LANA
Article Snippet: The full length and truncated versions of the N-terminal LANA (aa 1–340 (N), aa 100–340 (N6), aa 24–100 (N11), and aa 75–200 (N17)) were generated by PCR amplification from the pCDNA3-LANA and fused to GST expression cassette by ligation to pGEX-4T-1 (GE Healthcare). .. The luciferase reporter construct in the pGL2-ORF50p backbone containing the RTA promoter (3 kb sequence upstream of the RTA translation initiation codon) was prepared as described before and the luciferase reporter containing seven terminal repeats (TR) of KSHV genome, pGL3-7xTR, was a kind gift from Dr. Rolf Renne (University of Florida) .

Expressing:

Article Title: An N-Acyl Homoserine Lactone Synthase in the Ammonia-Oxidizing Bacterium Nitrosospira multiformis
Article Snippet: Amplified nmuI was cloned into pGEX-4T-1 (GE) as described by the manufacturer, and the resulting recombinant plasmid was termed pGEX-nmuI. .. We also created the LuxR homolog protein (NmuR) expression plasmid pET-R.

Article Title: The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA
Article Snippet: .. Expression vectors encoding for IQGAP1 C-terminal fragments fused with GST were generated by PCR and cloned into pGEX-4T-1 (GE Healthcare). .. C-terminal deletion mutants of GFP-IQGAP1-T1050AX2 were constructed as follows: ΔCC, a fragment corresponding to aa 1,563–1,657, was amplified by PCR and inserted into pEGFP-IQGAP1-T1050AX2 between an internal SwaI site (corresponding to aa position 1,347) and a SacII site of the 3′-end multiple cloning site fusing aa 1–1,374 with 1,563–1,657; ΔRGC, a fragment corresponding to aa 1–1,562, was amplified by PCR and cloned into the SalI–SacII sites of pEGFP-C3; ΔCC + RGC and pEGFP-IQGAP1-T1050AX2 were cleaved at SwaI (internal) and SmaI 3′-end multiple cloning sites and were self-ligated.

Article Title: A novel PIP2 binding of ?PKC and its contribution to the neurite induction ability 1
Article Snippet: .. The PCR product was subcloned into pTB701-GFP (described as BS340 in ) for the expression in mammalian cells and into pGEX-4T-1 (Amersham Pharmacia Biotech, Buckinghamshire, UK) for bacterial expression of a glutathione S -transferase (GST)-fusion protein, respectively. ..

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: Identification of dynein light chain road block-1 as a novel interaction partner with the human reduced folate carrier
Article Snippet: The full coding sequence of DYNLRB1 was inserted in frame into Bam HI/ Xho I sites of the bacterial expression vector pGEX-4T-1 (Invitrogen) to produce GST-DYNLRB1 fusion protein in Escherichia coli . .. Following the manufacturer's instructions, GST-DYNLRB1 fusion protein and GST were purified from BL-21 E. coli cells harboring recombinant pGEX-4T-1 and pGEX-4T-1, respectively, by using the Bulk GST Purification Module (Amersham Biosciences, Piscataway, NJ).

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: KSHV Reactivation from Latency Requires Pim-1 and Pim-3 Kinases to Inactivate the Latency-Associated Nuclear Antigen LANA
Article Snippet: .. The full length and truncated versions of the N-terminal LANA (aa 1–340 (N), aa 100–340 (N6), aa 24–100 (N11), and aa 75–200 (N17)) were generated by PCR amplification from the pCDNA3-LANA and fused to GST expression cassette by ligation to pGEX-4T-1 (GE Healthcare). .. The luciferase reporter construct in the pGL2-ORF50p backbone containing the RTA promoter (3 kb sequence upstream of the RTA translation initiation codon) was prepared as described before and the luciferase reporter containing seven terminal repeats (TR) of KSHV genome, pGL3-7xTR, was a kind gift from Dr. Rolf Renne (University of Florida) .

Transformation Assay:

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: Improved expression of recombinant fusion defensin gene plasmids packed with chitosan-derived nanoparticles and effect on antibacteria and mouse immunity
Article Snippet: .. E. coli (DH5α) cells transformed with pGB2B3 and pGEX-4T-1 were induced with isopropyl-β-D-thiogalactopyra-noside (IPTG) to express the fusion BD2/3 protein which was purified on a GST affinity column (Amersham Biosciences; GE Healthcare). .. The bioactivity of the fusion protein was measured by inhibition of 4 standard pathogen strains ( E. coli ATCC25922, S. aureus ATCC 26112, S. pneumoniae ATCC49619 and P. aeruginosa ATCC10211).

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 six constructs were then individually transformed into One Shot® Top10 (Invitrogen, CA) chemically competent E. coli for maintenance of the recombinant plasmids.

Article Title: Improved expression of recombinant fusion defensin gene plasmids packed with chitosan-derived nanoparticles and effect on antibacteria and mouse immunity
Article Snippet: .. Bioactivity assay in vitro of fusion BD2/3 protein expressed by E. coli E. coli (DH5α) cells transformed with pGB2B3 and pGEX-4T-1 were induced with isopropyl-β-D-thiogalactopyra-noside (IPTG) to express the fusion BD2/3 protein which was purified on a GST affinity column (Amersham Biosciences; GE Healthcare). .. The bioactivity of the fusion protein was measured by inhibition of 4 standard pathogen strains (E. coli ATCC25922, S. aureus ATCC 26112, S. pneumoniae ATCC49619 and P. aeruginosa ATCC10211).

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: Overexpression of CERKL, a gene responsible for retinitis pigmentosa in humans, protects cells from apoptosis induced by oxidative stress
Article Snippet: Purification of recombinant proteins To express the four CERKL isoforms as glutathione-S-transferase (GST) fusion proteins, we subcloned each variant in-frame into pGEX-4T-1 (Amersham Biosciences, Chalfont St. Giles, UK), between the XhoI and NotI sites. .. The constructs were transformed into BL21 Codon Plus E.coli cells (Stratagene) to avoid premature protein truncation due to the strongly biased codon usage of the CERKL human gene.

Derivative Assay:

Article Title: The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA
Article Snippet: Full-length ORF of exocyst subunits (Sec3, Sec8, Exo70, and Exo84) were subcloned into the yeast two-hybrid bait plasmid pB27 derived from the original pBTM116 plasmid ( ). .. Expression vectors encoding for IQGAP1 C-terminal fragments fused with GST were generated by PCR and cloned into pGEX-4T-1 (GE Healthcare).

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). .. 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.

Ligation:

Article Title: KSHV Reactivation from Latency Requires Pim-1 and Pim-3 Kinases to Inactivate the Latency-Associated Nuclear Antigen LANA
Article Snippet: .. The full length and truncated versions of the N-terminal LANA (aa 1–340 (N), aa 100–340 (N6), aa 24–100 (N11), and aa 75–200 (N17)) were generated by PCR amplification from the pCDNA3-LANA and fused to GST expression cassette by ligation to pGEX-4T-1 (GE Healthcare). .. The luciferase reporter construct in the pGL2-ORF50p backbone containing the RTA promoter (3 kb sequence upstream of the RTA translation initiation codon) was prepared as described before and the luciferase reporter containing seven terminal repeats (TR) of KSHV genome, pGL3-7xTR, was a kind gift from Dr. Rolf Renne (University of Florida) .

Generated:

Article Title: Characterization of dRFX2, a novel RFX family protein in Drosophila
Article Snippet: A fragment from −168 to −91 having base-substituted mutations was generated by the PCR method using p5′−168DPCNACAT ( ) as a template with primers −124MUT1P and SalI (−168) ( ). .. To create mutated derivatives in P-element vector backbones, fragments having various mutations in the region from −124 to −112 of the PCNA gene promoter were isolated from CAT plasmids by digestion with SalI (−168) and SacII (+24), and inserted between the XhoI (−607) and SacII (+24) sites of p5′−607DPCNA lacZ W8HS ( ). pACT-dRFX2202–589 , which was isolated by one-hybrid screening, was digested with XhoI and the isolated dRFX2 cDNA fragment was inserted into the SalI site of pGEX-4T-1 (Amersham Pharmacia Biotech) to create pGST-dRFX2202–480 , or the XhoI site of pUAST-HA to create pUAS-HA-dRFX2202–480 .

Article Title: Ablation of Cypher, a PDZ-LIM domain Z-line protein, causes a severe form of congenital myopathy
Article Snippet: .. Mutant PDZ domains were generated via PCR-based mutagenesis followed by subcloning into pGEX-4T-1 (Amersham Pharmacia Biotech) or pEGFP-C1 (CLONTECH). .. Each plasmid was sequenced to ensure that no PCR errors had been introduced.

Article Title: The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA
Article Snippet: .. Expression vectors encoding for IQGAP1 C-terminal fragments fused with GST were generated by PCR and cloned into pGEX-4T-1 (GE Healthcare). .. C-terminal deletion mutants of GFP-IQGAP1-T1050AX2 were constructed as follows: ΔCC, a fragment corresponding to aa 1,563–1,657, was amplified by PCR and inserted into pEGFP-IQGAP1-T1050AX2 between an internal SwaI site (corresponding to aa position 1,347) and a SacII site of the 3′-end multiple cloning site fusing aa 1–1,374 with 1,563–1,657; ΔRGC, a fragment corresponding to aa 1–1,562, was amplified by PCR and cloned into the SalI–SacII sites of pEGFP-C3; ΔCC + RGC and pEGFP-IQGAP1-T1050AX2 were cleaved at SwaI (internal) and SmaI 3′-end multiple cloning sites and were self-ligated.

Article Title: KSHV Reactivation from Latency Requires Pim-1 and Pim-3 Kinases to Inactivate the Latency-Associated Nuclear Antigen LANA
Article Snippet: .. The full length and truncated versions of the N-terminal LANA (aa 1–340 (N), aa 100–340 (N6), aa 24–100 (N11), and aa 75–200 (N17)) were generated by PCR amplification from the pCDNA3-LANA and fused to GST expression cassette by ligation to pGEX-4T-1 (GE Healthcare). .. The luciferase reporter construct in the pGL2-ORF50p backbone containing the RTA promoter (3 kb sequence upstream of the RTA translation initiation codon) was prepared as described before and the luciferase reporter containing seven terminal repeats (TR) of KSHV genome, pGL3-7xTR, was a kind gift from Dr. Rolf Renne (University of Florida) .

Inhibition:

Article Title: Improved expression of recombinant fusion defensin gene plasmids packed with chitosan-derived nanoparticles and effect on antibacteria and mouse immunity
Article Snippet: E. coli (DH5α) cells transformed with pGB2B3 and pGEX-4T-1 were induced with isopropyl-β-D-thiogalactopyra-noside (IPTG) to express the fusion BD2/3 protein which was purified on a GST affinity column (Amersham Biosciences; GE Healthcare). .. The bioactivity of the fusion protein was measured by inhibition of 4 standard pathogen strains ( E. coli ATCC25922, S. aureus ATCC 26112, S. pneumoniae ATCC49619 and P. aeruginosa ATCC10211).

Article Title: Improved expression of recombinant fusion defensin gene plasmids packed with chitosan-derived nanoparticles and effect on antibacteria and mouse immunity
Article Snippet: Bioactivity assay in vitro of fusion BD2/3 protein expressed by E. coli E. coli (DH5α) cells transformed with pGB2B3 and pGEX-4T-1 were induced with isopropyl-β-D-thiogalactopyra-noside (IPTG) to express the fusion BD2/3 protein which was purified on a GST affinity column (Amersham Biosciences; GE Healthcare). .. The bioactivity of the fusion protein was measured by inhibition of 4 standard pathogen strains (E. coli ATCC25922, S. aureus ATCC 26112, S. pneumoniae ATCC49619 and P. aeruginosa ATCC10211).

Polymerase Chain Reaction:

Article Title: Characterization of dRFX2, a novel RFX family protein in Drosophila
Article Snippet: A fragment from −168 to −91 having base-substituted mutations was generated by the PCR method using p5′−168DPCNACAT ( ) as a template with primers −124MUT1P and SalI (−168) ( ). .. To create mutated derivatives in P-element vector backbones, fragments having various mutations in the region from −124 to −112 of the PCNA gene promoter were isolated from CAT plasmids by digestion with SalI (−168) and SacII (+24), and inserted between the XhoI (−607) and SacII (+24) sites of p5′−607DPCNA lacZ W8HS ( ). pACT-dRFX2202–589 , which was isolated by one-hybrid screening, was digested with XhoI and the isolated dRFX2 cDNA fragment was inserted into the SalI site of pGEX-4T-1 (Amersham Pharmacia Biotech) to create pGST-dRFX2202–480 , or the XhoI site of pUAST-HA to create pUAS-HA-dRFX2202–480 .

Article Title: Ablation of Cypher, a PDZ-LIM domain Z-line protein, causes a severe form of congenital myopathy
Article Snippet: .. Mutant PDZ domains were generated via PCR-based mutagenesis followed by subcloning into pGEX-4T-1 (Amersham Pharmacia Biotech) or pEGFP-C1 (CLONTECH). .. Each plasmid was sequenced to ensure that no PCR errors had been introduced.

Article Title: The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA
Article Snippet: .. Expression vectors encoding for IQGAP1 C-terminal fragments fused with GST were generated by PCR and cloned into pGEX-4T-1 (GE Healthcare). .. C-terminal deletion mutants of GFP-IQGAP1-T1050AX2 were constructed as follows: ΔCC, a fragment corresponding to aa 1,563–1,657, was amplified by PCR and inserted into pEGFP-IQGAP1-T1050AX2 between an internal SwaI site (corresponding to aa position 1,347) and a SacII site of the 3′-end multiple cloning site fusing aa 1–1,374 with 1,563–1,657; ΔRGC, a fragment corresponding to aa 1–1,562, was amplified by PCR and cloned into the SalI–SacII sites of pEGFP-C3; ΔCC + RGC and pEGFP-IQGAP1-T1050AX2 were cleaved at SwaI (internal) and SmaI 3′-end multiple cloning sites and were self-ligated.

Article Title: A novel PIP2 binding of ?PKC and its contribution to the neurite induction ability 1
Article Snippet: .. The PCR product was subcloned into pTB701-GFP (described as BS340 in ) for the expression in mammalian cells and into pGEX-4T-1 (Amersham Pharmacia Biotech, Buckinghamshire, UK) for bacterial expression of a glutathione S -transferase (GST)-fusion protein, respectively. ..

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: KSHV Reactivation from Latency Requires Pim-1 and Pim-3 Kinases to Inactivate the Latency-Associated Nuclear Antigen LANA
Article Snippet: .. The full length and truncated versions of the N-terminal LANA (aa 1–340 (N), aa 100–340 (N6), aa 24–100 (N11), and aa 75–200 (N17)) were generated by PCR amplification from the pCDNA3-LANA and fused to GST expression cassette by ligation to pGEX-4T-1 (GE Healthcare). .. The luciferase reporter construct in the pGL2-ORF50p backbone containing the RTA promoter (3 kb sequence upstream of the RTA translation initiation codon) was prepared as described before and the luciferase reporter containing seven terminal repeats (TR) of KSHV genome, pGL3-7xTR, was a kind gift from Dr. Rolf Renne (University of Florida) .

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: 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). ..

Recombinant:

Article Title: An N-Acyl Homoserine Lactone Synthase in the Ammonia-Oxidizing Bacterium Nitrosospira multiformis
Article Snippet: .. Amplified nmuI was cloned into pGEX-4T-1 (GE) as described by the manufacturer, and the resulting recombinant plasmid was termed pGEX-nmuI. .. We also created the LuxR homolog protein (NmuR) expression plasmid pET-R.

Article Title: Identification of dynein light chain road block-1 as a novel interaction partner with the human reduced folate carrier
Article Snippet: .. Following the manufacturer's instructions, GST-DYNLRB1 fusion protein and GST were purified from BL-21 E. coli cells harboring recombinant pGEX-4T-1 and pGEX-4T-1, respectively, by using the Bulk GST Purification Module (Amersham Biosciences, Piscataway, NJ). .. The fusion protein and GST were separated by SDS-PAGE (8%), stained with Coomassie brilliant blue, and further used in GST pull-down assay.

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: 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: Overexpression of CERKL, a gene responsible for retinitis pigmentosa in humans, protects cells from apoptosis induced by oxidative stress
Article Snippet: .. Purification of recombinant proteins To express the four CERKL isoforms as glutathione-S-transferase (GST) fusion proteins, we subcloned each variant in-frame into pGEX-4T-1 (Amersham Biosciences, Chalfont St. Giles, UK), between the XhoI and NotI sites. .. The constructs were transformed into BL21 Codon Plus E.coli cells (Stratagene) to avoid premature protein truncation due to the strongly biased codon usage of the CERKL human gene.

Pull Down Assay:

Article Title: Identification of dynein light chain road block-1 as a novel interaction partner with the human reduced folate carrier
Article Snippet: Paragraph title: GST pull-down assay. ... Following the manufacturer's instructions, GST-DYNLRB1 fusion protein and GST were purified from BL-21 E. coli cells harboring recombinant pGEX-4T-1 and pGEX-4T-1, respectively, by using the Bulk GST Purification Module (Amersham Biosciences, Piscataway, NJ).

Mutagenesis:

Article Title: Ablation of Cypher, a PDZ-LIM domain Z-line protein, causes a severe form of congenital myopathy
Article Snippet: .. Mutant PDZ domains were generated via PCR-based mutagenesis followed by subcloning into pGEX-4T-1 (Amersham Pharmacia Biotech) or pEGFP-C1 (CLONTECH). .. Each plasmid was sequenced to ensure that no PCR errors had been introduced.

Article Title: A novel PIP2 binding of ?PKC and its contribution to the neurite induction ability 1
Article Snippet: The PCR product was subcloned into pTB701-GFP (described as BS340 in ) for the expression in mammalian cells and into pGEX-4T-1 (Amersham Pharmacia Biotech, Buckinghamshire, UK) for bacterial expression of a glutathione S -transferase (GST)-fusion protein, respectively. .. Anti-sense primers were 5-TTAGATCTTTCCTGGTCACAAGGGGA-3 for RRKK mutant II, 5-GAAGATCTTGACTTGGATCGGTCGTCTTC-3 for RRKK mutant, and 5-TTAGATCTTGGCCACTGTTGAT-3 for ΔRRKK, respectively.

Article Title: KSHV Reactivation from Latency Requires Pim-1 and Pim-3 Kinases to Inactivate the Latency-Associated Nuclear Antigen LANA
Article Snippet: The serine to arginine (SS205/206RR) mutations on LANA were generated using pCDNA3.1-LANA as a template by site-directed mutagenesis. .. The full length and truncated versions of the N-terminal LANA (aa 1–340 (N), aa 100–340 (N6), aa 24–100 (N11), and aa 75–200 (N17)) were generated by PCR amplification from the pCDNA3-LANA and fused to GST expression cassette by ligation to pGEX-4T-1 (GE Healthcare).

Isolation:

Article Title: Characterization of dRFX2, a novel RFX family protein in Drosophila
Article Snippet: .. To create mutated derivatives in P-element vector backbones, fragments having various mutations in the region from −124 to −112 of the PCNA gene promoter were isolated from CAT plasmids by digestion with SalI (−168) and SacII (+24), and inserted between the XhoI (−607) and SacII (+24) sites of p5′−607DPCNA lacZ W8HS ( ). pACT-dRFX2202–589 , which was isolated by one-hybrid screening, was digested with XhoI and the isolated dRFX2 cDNA fragment was inserted into the SalI site of pGEX-4T-1 (Amersham Pharmacia Biotech) to create pGST-dRFX2202–480 , or the XhoI site of pUAST-HA to create pUAS-HA-dRFX2202–480 . .. Full length of dRFX2 cDNA was isolated by 5′-RACE (Invitrogen) using total RNAs from larvae and the primer carrying the sequence, 5′-ACCGTAATATTCTAGAGG.

Article Title: An N-Acyl Homoserine Lactone Synthase in the Ammonia-Oxidizing Bacterium Nitrosospira multiformis
Article Snippet: Genomic DNA isolated from N. multiformis was used as the template for nmuI gene amplification with the primers 5′-CA GGATCC ATGCTTGCACAACATGGCA-3′ (5′ end) and 5′-CCG CTCGAG TCATGCGGCCTTCCTTTG-3′ (3′ end). .. Amplified nmuI was cloned into pGEX-4T-1 (GE) as described by the manufacturer, and the resulting recombinant plasmid was termed pGEX-nmuI.

Subcloning:

Article Title: Ablation of Cypher, a PDZ-LIM domain Z-line protein, causes a severe form of congenital myopathy
Article Snippet: .. Mutant PDZ domains were generated via PCR-based mutagenesis followed by subcloning into pGEX-4T-1 (Amersham Pharmacia Biotech) or pEGFP-C1 (CLONTECH). .. Each plasmid was sequenced to ensure that no PCR errors had been introduced.

Article Title: KSHV Reactivation from Latency Requires Pim-1 and Pim-3 Kinases to Inactivate the Latency-Associated Nuclear Antigen LANA
Article Snippet: The GST-tagged Pim-1 and Pim-1KD constructs were generated by PCR subcloning into the pGEX-6P-1 vector (GE Healthcare). pGEX-2T plasmid (GE Healthcare) was used to produce GST in control samples. .. The full length and truncated versions of the N-terminal LANA (aa 1–340 (N), aa 100–340 (N6), aa 24–100 (N11), and aa 75–200 (N17)) were generated by PCR amplification from the pCDNA3-LANA and fused to GST expression cassette by ligation to pGEX-4T-1 (GE Healthcare).

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. .. Proteins were extracted with B-PER (Pierce, Rockford, IL), purified with GST sepharose 4B (GE Healthcare) and eluted with 10 mM reduced glutathione (Sigma) in 50 mM Tris-HCl [pH 8.0] as previously described ( ).

Article Title: Identification of dynein light chain road block-1 as a novel interaction partner with the human reduced folate carrier
Article Snippet: .. Following the manufacturer's instructions, GST-DYNLRB1 fusion protein and GST were purified from BL-21 E. coli cells harboring recombinant pGEX-4T-1 and pGEX-4T-1, respectively, by using the Bulk GST Purification Module (Amersham Biosciences, Piscataway, NJ). .. The fusion protein and GST were separated by SDS-PAGE (8%), stained with Coomassie brilliant blue, and further used in GST pull-down assay.

Article Title: Improved expression of recombinant fusion defensin gene plasmids packed with chitosan-derived nanoparticles and effect on antibacteria and mouse immunity
Article Snippet: .. E. coli (DH5α) cells transformed with pGB2B3 and pGEX-4T-1 were induced with isopropyl-β-D-thiogalactopyra-noside (IPTG) to express the fusion BD2/3 protein which was purified on a GST affinity column (Amersham Biosciences; GE Healthcare). .. The bioactivity of the fusion protein was measured by inhibition of 4 standard pathogen strains ( E. coli ATCC25922, S. aureus ATCC 26112, S. pneumoniae ATCC49619 and P. aeruginosa ATCC10211).

Article Title: Arabidopsis FORGETTER1 mediates stress-induced chromatin memory through nucleosome remodeling
Article Snippet: .. Histone peptide binding assays The PHD domains of FGT1 (aa 667–757) and ING1 ( ) were subcloned into pGEX-4T-1 (Amersham Biosciences, Pittsburgh, PA), expressed in E. coli BL21 (DE3) as GST fusion proteins and purified using Glutathione affinity resins (Thermo Fisher Scientific, Waltham, MA). .. The histone peptide binding assay was performed as described ( ).

Article Title: Improved expression of recombinant fusion defensin gene plasmids packed with chitosan-derived nanoparticles and effect on antibacteria and mouse immunity
Article Snippet: .. Bioactivity assay in vitro of fusion BD2/3 protein expressed by E. coli E. coli (DH5α) cells transformed with pGB2B3 and pGEX-4T-1 were induced with isopropyl-β-D-thiogalactopyra-noside (IPTG) to express the fusion BD2/3 protein which was purified on a GST affinity column (Amersham Biosciences; GE Healthcare). .. The bioactivity of the fusion protein was measured by inhibition of 4 standard pathogen strains (E. coli ATCC25922, S. aureus ATCC 26112, S. pneumoniae ATCC49619 and P. aeruginosa ATCC10211).

Article Title: Overexpression of CERKL, a gene responsible for retinitis pigmentosa in humans, protects cells from apoptosis induced by oxidative stress
Article Snippet: .. Purification of recombinant proteins To express the four CERKL isoforms as glutathione-S-transferase (GST) fusion proteins, we subcloned each variant in-frame into pGEX-4T-1 (Amersham Biosciences, Chalfont St. Giles, UK), between the XhoI and NotI sites. .. The constructs were transformed into BL21 Codon Plus E.coli cells (Stratagene) to avoid premature protein truncation due to the strongly biased codon usage of the CERKL human gene.

Sequencing:

Article Title: Characterization of dRFX2, a novel RFX family protein in Drosophila
Article Snippet: To create mutated derivatives in P-element vector backbones, fragments having various mutations in the region from −124 to −112 of the PCNA gene promoter were isolated from CAT plasmids by digestion with SalI (−168) and SacII (+24), and inserted between the XhoI (−607) and SacII (+24) sites of p5′−607DPCNA lacZ W8HS ( ). pACT-dRFX2202–589 , which was isolated by one-hybrid screening, was digested with XhoI and the isolated dRFX2 cDNA fragment was inserted into the SalI site of pGEX-4T-1 (Amersham Pharmacia Biotech) to create pGST-dRFX2202–480 , or the XhoI site of pUAST-HA to create pUAS-HA-dRFX2202–480 . .. Full length of dRFX2 cDNA was isolated by 5′-RACE (Invitrogen) using total RNAs from larvae and the primer carrying the sequence, 5′-ACCGTAATATTCTAGAGG.

Article Title: The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA
Article Snippet: Alternatively, PCRs were performed with reverse primers, including the sequence for HA tag (5′-TACCCATACGATGTTCCAGATTACGCTTAA-3′), and fragments were cloned into pcDNA3.1 (Invitrogen). .. Expression vectors encoding for IQGAP1 C-terminal fragments fused with GST were generated by PCR and cloned into pGEX-4T-1 (GE Healthcare).

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: Identification of dynein light chain road block-1 as a novel interaction partner with the human reduced folate carrier
Article Snippet: The full coding sequence of DYNLRB1 was inserted in frame into Bam HI/ Xho I sites of the bacterial expression vector pGEX-4T-1 (Invitrogen) to produce GST-DYNLRB1 fusion protein in Escherichia coli . .. Following the manufacturer's instructions, GST-DYNLRB1 fusion protein and GST were purified from BL-21 E. coli cells harboring recombinant pGEX-4T-1 and pGEX-4T-1, respectively, by using the Bulk GST Purification Module (Amersham Biosciences, Piscataway, NJ).

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: Construction of the recombinant plasmids 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: KSHV Reactivation from Latency Requires Pim-1 and Pim-3 Kinases to Inactivate the Latency-Associated Nuclear Antigen LANA
Article Snippet: The full length and truncated versions of the N-terminal LANA (aa 1–340 (N), aa 100–340 (N6), aa 24–100 (N11), and aa 75–200 (N17)) were generated by PCR amplification from the pCDNA3-LANA and fused to GST expression cassette by ligation to pGEX-4T-1 (GE Healthcare). .. The luciferase reporter construct in the pGL2-ORF50p backbone containing the RTA promoter (3 kb sequence upstream of the RTA translation initiation codon) was prepared as described before and the luciferase reporter containing seven terminal repeats (TR) of KSHV genome, pGL3-7xTR, was a kind gift from Dr. Rolf Renne (University of Florida) .

Positron Emission Tomography:

Article Title: An N-Acyl Homoserine Lactone Synthase in the Ammonia-Oxidizing Bacterium Nitrosospira multiformis
Article Snippet: Amplified nmuI was cloned into pGEX-4T-1 (GE) as described by the manufacturer, and the resulting recombinant plasmid was termed pGEX-nmuI. .. We also created the LuxR homolog protein (NmuR) expression plasmid pET-R.

Affinity Column:

Article Title: Improved expression of recombinant fusion defensin gene plasmids packed with chitosan-derived nanoparticles and effect on antibacteria and mouse immunity
Article Snippet: .. E. coli (DH5α) cells transformed with pGB2B3 and pGEX-4T-1 were induced with isopropyl-β-D-thiogalactopyra-noside (IPTG) to express the fusion BD2/3 protein which was purified on a GST affinity column (Amersham Biosciences; GE Healthcare). .. The bioactivity of the fusion protein was measured by inhibition of 4 standard pathogen strains ( E. coli ATCC25922, S. aureus ATCC 26112, S. pneumoniae ATCC49619 and P. aeruginosa ATCC10211).

Article Title: Improved expression of recombinant fusion defensin gene plasmids packed with chitosan-derived nanoparticles and effect on antibacteria and mouse immunity
Article Snippet: .. Bioactivity assay in vitro of fusion BD2/3 protein expressed by E. coli E. coli (DH5α) cells transformed with pGB2B3 and pGEX-4T-1 were induced with isopropyl-β-D-thiogalactopyra-noside (IPTG) to express the fusion BD2/3 protein which was purified on a GST affinity column (Amersham Biosciences; GE Healthcare). .. The bioactivity of the fusion protein was measured by inhibition of 4 standard pathogen strains (E. coli ATCC25922, S. aureus ATCC 26112, S. pneumoniae ATCC49619 and P. aeruginosa ATCC10211).

Staining:

Article Title: Identification of dynein light chain road block-1 as a novel interaction partner with the human reduced folate carrier
Article Snippet: Following the manufacturer's instructions, GST-DYNLRB1 fusion protein and GST were purified from BL-21 E. coli cells harboring recombinant pGEX-4T-1 and pGEX-4T-1, respectively, by using the Bulk GST Purification Module (Amersham Biosciences, Piscataway, NJ). .. The fusion protein and GST were separated by SDS-PAGE (8%), stained with Coomassie brilliant blue, and further used in GST pull-down assay.

Chloramphenicol Acetyltransferase Assay:

Article Title: Characterization of dRFX2, a novel RFX family protein in Drosophila
Article Snippet: .. To create mutated derivatives in P-element vector backbones, fragments having various mutations in the region from −124 to −112 of the PCNA gene promoter were isolated from CAT plasmids by digestion with SalI (−168) and SacII (+24), and inserted between the XhoI (−607) and SacII (+24) sites of p5′−607DPCNA lacZ W8HS ( ). pACT-dRFX2202–589 , which was isolated by one-hybrid screening, was digested with XhoI and the isolated dRFX2 cDNA fragment was inserted into the SalI site of pGEX-4T-1 (Amersham Pharmacia Biotech) to create pGST-dRFX2202–480 , or the XhoI site of pUAST-HA to create pUAS-HA-dRFX2202–480 . .. Full length of dRFX2 cDNA was isolated by 5′-RACE (Invitrogen) using total RNAs from larvae and the primer carrying the sequence, 5′-ACCGTAATATTCTAGAGG.

SDS Page:

Article Title: Identification of dynein light chain road block-1 as a novel interaction partner with the human reduced folate carrier
Article Snippet: Following the manufacturer's instructions, GST-DYNLRB1 fusion protein and GST were purified from BL-21 E. coli cells harboring recombinant pGEX-4T-1 and pGEX-4T-1, respectively, by using the Bulk GST Purification Module (Amersham Biosciences, Piscataway, NJ). .. The fusion protein and GST were separated by SDS-PAGE (8%), stained with Coomassie brilliant blue, and further used in GST pull-down assay.

Article Title: Arabidopsis FORGETTER1 mediates stress-induced chromatin memory through nucleosome remodeling
Article Snippet: Histone peptide binding assays The PHD domains of FGT1 (aa 667–757) and ING1 ( ) were subcloned into pGEX-4T-1 (Amersham Biosciences, Pittsburgh, PA), expressed in E. coli BL21 (DE3) as GST fusion proteins and purified using Glutathione affinity resins (Thermo Fisher Scientific, Waltham, MA). .. After incubation with Streptavidin Dynabeads (Thermo Fisher Scientific) and extensive washing with TBST, bound proteins were analyzed by SDS-PAGE and immunoblotting with anti-GST antibodies (Merck-Millipore).

Plasmid Preparation:

Article Title: Characterization of dRFX2, a novel RFX family protein in Drosophila
Article Snippet: .. To create mutated derivatives in P-element vector backbones, fragments having various mutations in the region from −124 to −112 of the PCNA gene promoter were isolated from CAT plasmids by digestion with SalI (−168) and SacII (+24), and inserted between the XhoI (−607) and SacII (+24) sites of p5′−607DPCNA lacZ W8HS ( ). pACT-dRFX2202–589 , which was isolated by one-hybrid screening, was digested with XhoI and the isolated dRFX2 cDNA fragment was inserted into the SalI site of pGEX-4T-1 (Amersham Pharmacia Biotech) to create pGST-dRFX2202–480 , or the XhoI site of pUAST-HA to create pUAS-HA-dRFX2202–480 . .. Full length of dRFX2 cDNA was isolated by 5′-RACE (Invitrogen) using total RNAs from larvae and the primer carrying the sequence, 5′-ACCGTAATATTCTAGAGG.

Article Title: An N-Acyl Homoserine Lactone Synthase in the Ammonia-Oxidizing Bacterium Nitrosospira multiformis
Article Snippet: .. Amplified nmuI was cloned into pGEX-4T-1 (GE) as described by the manufacturer, and the resulting recombinant plasmid was termed pGEX-nmuI. .. We also created the LuxR homolog protein (NmuR) expression plasmid pET-R.

Article Title: Ablation of Cypher, a PDZ-LIM domain Z-line protein, causes a severe form of congenital myopathy
Article Snippet: Paragraph title: Plasmid construction ... Mutant PDZ domains were generated via PCR-based mutagenesis followed by subcloning into pGEX-4T-1 (Amersham Pharmacia Biotech) or pEGFP-C1 (CLONTECH).

Article Title: The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA
Article Snippet: Paragraph title: Plasmid constructions ... Expression vectors encoding for IQGAP1 C-terminal fragments fused with GST were generated by PCR and cloned into pGEX-4T-1 (GE Healthcare).

Article Title: A novel PIP2 binding of ?PKC and its contribution to the neurite induction ability 1
Article Snippet: Constructs of plasmids encoding GFP-fused εPKC and mutants The plasmid encoding εPKC having GFP at its C terminus [full length εPKC (εFL)–GFP] was described previously ( ). .. The PCR product was subcloned into pTB701-GFP (described as BS340 in ) for the expression in mammalian cells and into pGEX-4T-1 (Amersham Pharmacia Biotech, Buckinghamshire, UK) for bacterial expression of a glutathione S -transferase (GST)-fusion protein, respectively.

Article Title: Identification of dynein light chain road block-1 as a novel interaction partner with the human reduced folate carrier
Article Snippet: The full coding sequence of DYNLRB1 was inserted in frame into Bam HI/ Xho I sites of the bacterial expression vector pGEX-4T-1 (Invitrogen) to produce GST-DYNLRB1 fusion protein in Escherichia coli . .. Following the manufacturer's instructions, GST-DYNLRB1 fusion protein and GST were purified from BL-21 E. coli cells harboring recombinant pGEX-4T-1 and pGEX-4T-1, respectively, by using the Bulk GST Purification Module (Amersham Biosciences, Piscataway, NJ).

Article Title: KSHV Reactivation from Latency Requires Pim-1 and Pim-3 Kinases to Inactivate the Latency-Associated Nuclear Antigen LANA
Article Snippet: The GST-tagged Pim-1 and Pim-1KD constructs were generated by PCR subcloning into the pGEX-6P-1 vector (GE Healthcare). pGEX-2T plasmid (GE Healthcare) was used to produce GST in control samples. .. The full length and truncated versions of the N-terminal LANA (aa 1–340 (N), aa 100–340 (N6), aa 24–100 (N11), and aa 75–200 (N17)) were generated by PCR amplification from the pCDNA3-LANA and fused to GST expression cassette by ligation to pGEX-4T-1 (GE Healthcare).

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).

Binding Assay:

Article Title: Arabidopsis FORGETTER1 mediates stress-induced chromatin memory through nucleosome remodeling
Article Snippet: .. Histone peptide binding assays The PHD domains of FGT1 (aa 667–757) and ING1 ( ) were subcloned into pGEX-4T-1 (Amersham Biosciences, Pittsburgh, PA), expressed in E. coli BL21 (DE3) as GST fusion proteins and purified using Glutathione affinity resins (Thermo Fisher Scientific, Waltham, MA). .. The histone peptide binding assay was performed as described ( ).

In Vitro:

Article Title: Improved expression of recombinant fusion defensin gene plasmids packed with chitosan-derived nanoparticles and effect on antibacteria and mouse immunity
Article Snippet: Paragraph title: Bioactivity assay in vitro of fusion BD2/3 protein expressed by E. coli ... E. coli (DH5α) cells transformed with pGB2B3 and pGEX-4T-1 were induced with isopropyl-β-D-thiogalactopyra-noside (IPTG) to express the fusion BD2/3 protein which was purified on a GST affinity column (Amersham Biosciences; GE Healthcare).

Article Title: Improved expression of recombinant fusion defensin gene plasmids packed with chitosan-derived nanoparticles and effect on antibacteria and mouse immunity
Article Snippet: .. Bioactivity assay in vitro of fusion BD2/3 protein expressed by E. coli E. coli (DH5α) cells transformed with pGB2B3 and pGEX-4T-1 were induced with isopropyl-β-D-thiogalactopyra-noside (IPTG) to express the fusion BD2/3 protein which was purified on a GST affinity column (Amersham Biosciences; GE Healthcare). .. The bioactivity of the fusion protein was measured by inhibition of 4 standard pathogen strains (E. coli ATCC25922, S. aureus ATCC 26112, S. pneumoniae ATCC49619 and P. aeruginosa ATCC10211).

Produced:

Article Title: A novel PIP2 binding of ?PKC and its contribution to the neurite induction ability 1
Article Snippet: A cDNA fragments encoding εRD with Bgl II site was produced by a PCR with cDNA for rat εPKC as the template. .. The PCR product was subcloned into pTB701-GFP (described as BS340 in ) for the expression in mammalian cells and into pGEX-4T-1 (Amersham Pharmacia Biotech, Buckinghamshire, UK) for bacterial expression of a glutathione S -transferase (GST)-fusion protein, respectively.

Article Title: Overexpression of CERKL, a gene responsible for retinitis pigmentosa in humans, protects cells from apoptosis induced by oxidative stress
Article Snippet: Purification of recombinant proteins To express the four CERKL isoforms as glutathione-S-transferase (GST) fusion proteins, we subcloned each variant in-frame into pGEX-4T-1 (Amersham Biosciences, Chalfont St. Giles, UK), between the XhoI and NotI sites. .. Recombinant proteins were produced in 500 ml cultures induced by the addition of 0.5 mM Isopropyl-β-D-thiogalactopyranoside (IPTG).

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). .. 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.

Variant Assay:

Article Title: Overexpression of CERKL, a gene responsible for retinitis pigmentosa in humans, protects cells from apoptosis induced by oxidative stress
Article Snippet: .. Purification of recombinant proteins To express the four CERKL isoforms as glutathione-S-transferase (GST) fusion proteins, we subcloned each variant in-frame into pGEX-4T-1 (Amersham Biosciences, Chalfont St. Giles, UK), between the XhoI and NotI sites. .. The constructs were transformed into BL21 Codon Plus E.coli cells (Stratagene) to avoid premature protein truncation due to the strongly biased codon usage of the CERKL human gene.

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    GE Healthcare pgex 4t 1
    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 <t>pGEX-4T-1.</t> 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.
    Pgex 4t 1, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 288 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pgex 4t 1/product/GE Healthcare
    Average 99 stars, based on 288 article reviews
    Price from $9.99 to $1999.99
    pgex 4t 1 - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    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 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gst fusion proteins/product/GE Healthcare
    Average 99 stars, based on 35 article reviews
    Price from $9.99 to $1999.99
    gst fusion proteins - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    91
    GE Healthcare pgex 4t 1 expression plasmid
    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, <t>pGEX-4T-1</t> 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.
    Pgex 4t 1 Expression Plasmid, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pgex 4t 1 expression plasmid/product/GE Healthcare
    Average 91 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    pgex 4t 1 expression plasmid - by Bioz Stars, 2020-02
    91/100 stars
      Buy from Supplier

    Image Search Results


    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.

    Journal: PLoS Neglected Tropical Diseases

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

    doi: 10.1371/journal.pntd.0002788

    Figure Lengend 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.

    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.

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

    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.

    Journal: BMC Veterinary Research

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

    doi: 10.1186/1746-6148-10-115

    Figure Lengend 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.

    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.

    Techniques: Clone Assay, 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: Plasmids and peptides 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

    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