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.
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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 "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

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 "Profiling of Viral Proteins Expressed from the Genomic RNA of Japanese Encephalitis Virus Using a Panel of 15 Region-Specific Polyclonal Rabbit Antisera: Implications for Viral Gene Expression"

Article Title: Profiling of Viral Proteins Expressed from the Genomic RNA of Japanese Encephalitis Virus Using a Panel of 15 Region-Specific Polyclonal Rabbit Antisera: Implications for Viral Gene Expression

Journal: PLoS ONE

doi: 10.1371/journal.pone.0124318

Production of five GST fusion proteins, each containing a non-hydrophobic region from the JEV structural protein-coding region. GST fusion proteins were expressed from pGex-4T-1 vector in E . coli and purified from bacterial lysates by affinity chromatography using glutathione-Sepharose. About 5–10 μg of each purified protein was resolved on a SDS-polyacrylamide gel and stained with Coomassie blue. The five GST fusion proteins we generated are: GST-C ( A ), GST-pr ( B ), GST-M ( C ), GST-E N-term ( D ), and GST-E C-term ( E ). Free GST protein was used as control. Molecular weight markers are indicated in kDa on the left side of each panel, and GST and GST fusion proteins are marked on the right side. The predicted molecular weights are indicated at the bottom of each panel.
Figure Legend Snippet: Production of five GST fusion proteins, each containing a non-hydrophobic region from the JEV structural protein-coding region. GST fusion proteins were expressed from pGex-4T-1 vector in E . coli and purified from bacterial lysates by affinity chromatography using glutathione-Sepharose. About 5–10 μg of each purified protein was resolved on a SDS-polyacrylamide gel and stained with Coomassie blue. The five GST fusion proteins we generated are: GST-C ( A ), GST-pr ( B ), GST-M ( C ), GST-E N-term ( D ), and GST-E C-term ( E ). Free GST protein was used as control. Molecular weight markers are indicated in kDa on the left side of each panel, and GST and GST fusion proteins are marked on the right side. The predicted molecular weights are indicated at the bottom of each panel.

Techniques Used: Plasmid Preparation, Purification, Affinity Chromatography, Staining, Generated, Molecular Weight

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 "N-terminal Domain of the Spike Protein of Porcine Epidemic Diarrhea Virus as a New Candidate Molecule for a Mucosal Vaccine"

Article Title: N-terminal Domain of the Spike Protein of Porcine Epidemic Diarrhea Virus as a New Candidate Molecule for a Mucosal Vaccine

Journal: Immune Network

doi: 10.4110/in.2018.18.e21

Expression of the NTD 231–501 of the PEDV S1 protein in E. coli . (A) The gene region encoding NTD 231–501 was cloned into the pGEX 4T-1 vector containing a GST tag. (B) Recombinant NTD 231–501 of the PEDV S1 protein was expressed by isopropyl β-D-1-thiogalactopyranoside induction and confirmed by Western blot analysis using an anti-GST Ab. (C) Recombinant NTD 231–501 of the PEDV S1 protein was purified by glutathione Sepharose chromatography.
Figure Legend Snippet: Expression of the NTD 231–501 of the PEDV S1 protein in E. coli . (A) The gene region encoding NTD 231–501 was cloned into the pGEX 4T-1 vector containing a GST tag. (B) Recombinant NTD 231–501 of the PEDV S1 protein was expressed by isopropyl β-D-1-thiogalactopyranoside induction and confirmed by Western blot analysis using an anti-GST Ab. (C) Recombinant NTD 231–501 of the PEDV S1 protein was purified by glutathione Sepharose chromatography.

Techniques Used: Expressing, Clone Assay, Plasmid Preparation, Recombinant, Western Blot, Purification, Chromatography

8) Product Images from "N-terminal Domain of the Spike Protein of Porcine Epidemic Diarrhea Virus as a New Candidate Molecule for a Mucosal Vaccine"

Article Title: N-terminal Domain of the Spike Protein of Porcine Epidemic Diarrhea Virus as a New Candidate Molecule for a Mucosal Vaccine

Journal: Immune Network

doi: 10.4110/in.2018.18.e21

Expression of the NTD 231–501 of the PEDV S1 protein in E. coli . (A) The gene region encoding NTD 231–501 was cloned into the pGEX 4T-1 vector containing a GST tag. (B) Recombinant NTD 231–501 of the PEDV S1 protein was expressed by isopropyl β-D-1-thiogalactopyranoside induction and confirmed by Western blot analysis using an anti-GST Ab. (C) Recombinant NTD 231–501 of the PEDV S1 protein was purified by glutathione Sepharose chromatography.
Figure Legend Snippet: Expression of the NTD 231–501 of the PEDV S1 protein in E. coli . (A) The gene region encoding NTD 231–501 was cloned into the pGEX 4T-1 vector containing a GST tag. (B) Recombinant NTD 231–501 of the PEDV S1 protein was expressed by isopropyl β-D-1-thiogalactopyranoside induction and confirmed by Western blot analysis using an anti-GST Ab. (C) Recombinant NTD 231–501 of the PEDV S1 protein was purified by glutathione Sepharose chromatography.

Techniques Used: Expressing, Clone Assay, Plasmid Preparation, Recombinant, Western Blot, Purification, Chromatography

9) Product Images from "High Expression of Water-Soluble Recombinant Antigenic Domains of Toxoplasma gondii Secretory Organelles"

Article Title: High Expression of Water-Soluble Recombinant Antigenic Domains of Toxoplasma gondii Secretory Organelles

Journal: The Korean Journal of Parasitology

doi: 10.3347/kjp.2014.52.4.367

Production of GST-ROP2 fusion protein. (A) Design of fragmentation of cDNA of ROP2. Eight fragments of ROP2 were cloned as
Figure Legend Snippet: Production of GST-ROP2 fusion protein. (A) Design of fragmentation of cDNA of ROP2. Eight fragments of ROP2 were cloned as "A", full sequence of ROP2 without signal sequence; "B", 1/2 Nt sequence of ROP2; "C", 1/2 Ct containing kinase domain; "D", 1/4 Nt; "E", middle 1/4 Ct; "G", 1/4Ct; "H", 1/2 Nt of kinase domain; and "I", 1/2 Ct of kinase domain. (B) Expression of recombinant ROP2 antigens. All recombinant proteins were well induced and tested by western blot with anti-GST antibody. (C) Antigenicity of recombinant ROP2 antigens. The antigenicity was tested by western blot against patient serum. (D) Solubility of recombinant ROP2 antigens. Solubility of rGST-ROP2 324-561 induced was confirmed by western blot with anti-GST antibody. (E) Expression of recombinant linker ROP2 antigens. "L", lysate of BL21 (DE3) pLysS E. coli transformed with pGEX-4T-1/GRA2 31-71 ; "R2", that with pGEX-4T-1/ROP2 324-561 ; and "LR", that with pGEX-4T-1/GRA2 31-71 -ROP2 324-561 . The expression was confirmed by western blot with anti-GST antibody. (F) Antigenicity of recombinant linker ROP2 antigens. It was tested by western blot against patient serum. (G) Solubility of recombinant linker ROP2 antigens. Soluble and insoluble fractions of rGST-GRA2 31-71 -ROP2 324-561 protein were tested by western blot.

Techniques Used: Clone Assay, Sequencing, Expressing, Recombinant, Western Blot, Solubility, Transformation Assay

Production of GST-MIC2 fusion protein. (A) Design of fragmentation of cDNA of MIC2. Ten fragments of MIC2 were cloned as
Figure Legend Snippet: Production of GST-MIC2 fusion protein. (A) Design of fragmentation of cDNA of MIC2. Ten fragments of MIC2 were cloned as "F", full sequence of MIC2; "A", full sequence without Ct transmembrane region; "B", 2/3 Nt; "C", 2/3 Ct; "D", 1/3 Nt; "E", middle 1/3; "G", 1/3 Ct; "H", half Nt of D; "I", half Ct of D; and "J", middle 1/3 of D. (B) Expression of recombinant MIC2 antigens. All recombinant proteins were well induced and tested by western blot against anti-GST antibody. (C) Antigenicity of recombinant MIC2 antigens. The antigenicity of GST-MIC2 was tested by western blot against patient serum. (D) Solubility of recombinant MIC2 antigens. Total lysate, soluble, and insoluble fractions of rGST-MIC2 1-284 were detected against GST antibody. (E) Expression of recombinant linker MIC2 antigens. BL21 (DE3) pLysS E. coli containing GST and GST linker MIC2 1-284 recombinant plasmids were induced; "L", lysate transformed with pGEX-4T-1/GRA2 31-71 ; "M2", that with pGEX-4T-1/MIC2 1-284 ; and "LM", that with pGEX-4T-1/GRA2 31-71 -MIC2 1-284 . The expression was confirmed against anti-GST antibody. (F) Antigenicity of recombinant linker MIC2 antigens. It was tested against patient serum. (G) Solubility of recombinant linker MIC2 antigens. The soluble and insoluble fractions of rGST-GRA2 31-71 -MIC2 1-284 protein were tested by western blot.

Techniques Used: Clone Assay, Sequencing, Expressing, Recombinant, Western Blot, Solubility, Transformation Assay

10) Product Images from "High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development"

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

Journal: Microbial Cell Factories

doi: 10.1186/1475-2859-10-56

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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used:

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.
Figure Legend 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.

Techniques Used:

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.
Figure Legend 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.

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

11) Product Images from "Thromboxane A2 modulates cisplatin-induced apoptosis through a Siva1-dependent mechanism"

Article Title: Thromboxane A2 modulates cisplatin-induced apoptosis through a Siva1-dependent mechanism

Journal: Cell Death and Differentiation

doi: 10.1038/cdd.2012.11

Siva1 interacts with the C-tail and intra-cellular loops (ICL) of TP β through its domain spanning amino acids 138 through 175. ( a ) Various fragments of the TP β C-tail were cloned into the pGEX-4T-1 vector to generate GST fusion proteins.
Figure Legend Snippet: Siva1 interacts with the C-tail and intra-cellular loops (ICL) of TP β through its domain spanning amino acids 138 through 175. ( a ) Various fragments of the TP β C-tail were cloned into the pGEX-4T-1 vector to generate GST fusion proteins.

Techniques Used: Clone Assay, Plasmid Preparation

12) 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

13) 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

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Clone Assay:

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Article Title: N-terminal Domain of the Spike Protein of Porcine Epidemic Diarrhea Virus as a New Candidate Molecule for a Mucosal Vaccine
Article Snippet: .. The amplified NTD231–501 gene was cloned into the pGEX 4T-1 expression vector (GE Healthcare Life Sciences, Buckinghamshire, UK) or the pET SUMO vector (Thermo Fisher Scientific, Waltham, VA, USA). .. The recombinant NTD231–501 protein expressed in the Escherichia coli (E. coli ) BL21 (DE3) strain was purified using a glutathione- or Ni-NTA-based affinity purification system (GE Healthcare Life Sciences and Thermo Fischer Scientific, respectively) according to the manufacturers' instructions.

Article Title: Improved expression of recombinant fusion defensin gene plasmids packed with chitosan-derived nanoparticles and effect on antibacteria and mouse immunity
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Article Title: Improved expression of recombinant fusion defensin gene plasmids packed with chitosan-derived nanoparticles and effect on antibacteria and mouse immunity
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Article Title: High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development
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Article Title: High yield production of pigeon circovirus capsid protein in the E. coli by evaluating the key parameters needed for protein expression
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Article Snippet: The full-length open reading frame of Siva1 was cloned by RT-PCR using template RNA extracted from HeLa cells. .. Generation of the TP constructs in the pGEX-4T-1 vector (GE Healthcare) was described previously.

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: Profiling of Viral Proteins Expressed from the Genomic RNA of Japanese Encephalitis Virus Using a Panel of 15 Region-Specific Polyclonal Rabbit Antisera: Implications for Viral Gene Expression
Article Snippet: In all 16 plasmids, a defined region of the JEV ORF was first amplified by PCR using the full-length infectious cDNA molecular clone of JEV CNU/LP2 (pBACSP6 /JVFLx/XbaI [ ]) as a template and the appropriate pair of primers listed in (note that primer names indicate the target viral protein-coding region, followed by the orientation of each primer, forward (f) or reverse (r)). .. Next, the resulting PCR amplicons were individually ligated in-frame to the 3'-end of GST coding sequence in the pGex-4T-1 vector (GE Healthcare, Piscataway, NJ) using either the Eco RI and Xho I sites (for the first 14 PCR amplicons except NS5N-term and NS5C-term ) or the Bam HI and Xho I sites (for NS5N-term and NS5C-term ).

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: N-terminal Domain of the Spike Protein of Porcine Epidemic Diarrhea Virus as a New Candidate Molecule for a Mucosal Vaccine
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Article Title: High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development
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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: N-terminal Domain of the Spike Protein of Porcine Epidemic Diarrhea Virus as a New Candidate Molecule for a Mucosal Vaccine
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Article Title: Improved expression of recombinant fusion defensin gene plasmids packed with chitosan-derived nanoparticles and effect on antibacteria and mouse immunity
Article Snippet: Construction of prokaryotic expression plasmid of fusion gene of BD2/3 To construct a fusion gene of BD2/3 (GenBank: AF071216, AF301470), six oligodeoxynucleotide fragments were designed and synthesized by Sangon Biotech Co., Ltd., Shanghai, China ( ). .. 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.

Article Title: Improved expression of recombinant fusion defensin gene plasmids packed with chitosan-derived nanoparticles and effect on antibacteria and mouse immunity
Article Snippet: To construct a fusion gene of BD2/3 (GenBank: , ), six oligodeoxynucleotide fragments were designed and synthesized by Sangon Biotech Co., Ltd., Shanghai, China ( ). .. 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.

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: Profiling of Viral Proteins Expressed from the Genomic RNA of Japanese Encephalitis Virus Using a Panel of 15 Region-Specific Polyclonal Rabbit Antisera: Implications for Viral Gene Expression
Article Snippet: Plasmid construction A total of 16 bacterial expression plasmids were constructed, each of which was used to express a small non-hydrophobic region of the JEV polyprotein as a glutathione S -transferase (GST) fusion protein. .. Next, the resulting PCR amplicons were individually ligated in-frame to the 3'-end of GST coding sequence in the pGex-4T-1 vector (GE Healthcare, Piscataway, NJ) using either the Eco RI and Xho I sites (for the first 14 PCR amplicons except NS5N-term and NS5C-term ) or the Bam HI and Xho I sites (for NS5N-term and NS5C-term ).

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: N-terminal Domain of the Spike Protein of Porcine Epidemic Diarrhea Virus as a New Candidate Molecule for a Mucosal Vaccine
Article Snippet: The amplified NTD231–501 gene was cloned into the pGEX 4T-1 expression vector (GE Healthcare Life Sciences, Buckinghamshire, UK) or the pET SUMO vector (Thermo Fisher Scientific, Waltham, VA, USA). .. The recombinant NTD231–501 protein containing a glutathione S-transferase (GST) tag was expressed from the pGEX 4T-1 vector, and the recombinant NTD231–501 protein without a GST tag was expressed from the pSUMO vector; these constructs were used as the immunization and coating Ags, respectively, in subsequent experiments.

Article Title: Improved expression of recombinant fusion defensin gene plasmids packed with chitosan-derived nanoparticles and effect on antibacteria and mouse immunity
Article Snippet: Construction of prokaryotic expression plasmid of fusion gene of BD2/3 To construct a fusion gene of BD2/3 (GenBank: AF071216, AF301470), six oligodeoxynucleotide fragments were designed and synthesized by Sangon Biotech Co., Ltd., Shanghai, China ( ). .. 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.

Article Title: Improved expression of recombinant fusion defensin gene plasmids packed with chitosan-derived nanoparticles and effect on antibacteria and mouse immunity
Article Snippet: To construct a fusion gene of BD2/3 (GenBank: , ), six oligodeoxynucleotide fragments were designed and synthesized by Sangon Biotech Co., Ltd., Shanghai, China ( ). .. 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.

Article Title: High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development
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). .. This construct carries the VP1 gene flanked by the N-terminal glutathione-S-transferase (GST) tag and will express a GST-VP1 fusion protein.

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: Thromboxane A2 modulates cisplatin-induced apoptosis through a Siva1-dependent mechanism
Article Snippet: .. Generation of the TP constructs in the pGEX-4T-1 vector (GE Healthcare) was described previously. .. Myc-Mdm2, Myc-TRAF2 and Myc-XIAP were all cloned by RT-PCR from HeLa cells mRNA and inserted into the pcDNA3 expression vector (Invitrogen).

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.

Expressing:

Article Title: Profiling of Viral Proteins Expressed from the Genomic RNA of Japanese Encephalitis Virus Using a Panel of 15 Region-Specific Polyclonal Rabbit Antisera: Implications for Viral Gene Expression
Article Snippet: Plasmid construction A total of 16 bacterial expression plasmids were constructed, each of which was used to express a small non-hydrophobic region of the JEV polyprotein as a glutathione S -transferase (GST) fusion protein. .. Next, the resulting PCR amplicons were individually ligated in-frame to the 3'-end of GST coding sequence in the pGex-4T-1 vector (GE Healthcare, Piscataway, NJ) using either the Eco RI and Xho I sites (for the first 14 PCR amplicons except NS5N-term and NS5C-term ) or the Bam HI and Xho I sites (for NS5N-term and NS5C-term ).

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: N-terminal Domain of the Spike Protein of Porcine Epidemic Diarrhea Virus as a New Candidate Molecule for a Mucosal Vaccine
Article Snippet: .. The amplified NTD231–501 gene was cloned into the pGEX 4T-1 expression vector (GE Healthcare Life Sciences, Buckinghamshire, UK) or the pET SUMO vector (Thermo Fisher Scientific, Waltham, VA, USA). .. The recombinant NTD231–501 protein expressed in the Escherichia coli (E. coli ) BL21 (DE3) strain was purified using a glutathione- or Ni-NTA-based affinity purification system (GE Healthcare Life Sciences and Thermo Fischer Scientific, respectively) according to the manufacturers' instructions.

Article Title: Improved expression of recombinant fusion defensin gene plasmids packed with chitosan-derived nanoparticles and effect on antibacteria and mouse immunity
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. .. Sequence of the resulting plasmid was confirmed by double restriction enzyme digestions and sequencing, and designated as pGB2B3.

Article Title: Improved expression of recombinant fusion defensin gene plasmids packed with chitosan-derived nanoparticles and effect on antibacteria and mouse immunity
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. .. Sequence of the resulting plasmid was confirmed by double restriction enzyme digestions and sequencing, and designated as pGB2B3.

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: N-terminal Domain of the Spike Protein of Porcine Epidemic Diarrhea Virus as a New Candidate Molecule for a Mucosal Vaccine
Article Snippet: .. The NTD231–501 gene of the PEDV S1 protein was inserted into the pGEX 4T-1 expression vector containing a thrombin recognition site for cleaving the target protein. .. After expressing the construct in E. coli (BL21 Star (DE3)), the recombinant NTD231–501 protein yield was 20 mg/L.

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: Thromboxane A2 modulates cisplatin-induced apoptosis through a Siva1-dependent mechanism
Article Snippet: The PCR products were inserted into either the pcDNA3 expression vector (Life Technologies) to yield the Siva1-HA and Siva1ΔC-HA constructs or into the pRSETa expression vector (Invitrogen) to yield the 6 × His-Siva1-HA, 6 × His-Siva1ΔC-HA and 6 × His-Siva1ΔΔC-HA constructs. .. Generation of the TP constructs in the pGEX-4T-1 vector (GE Healthcare) was described previously.

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.

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: High Expression of Water-Soluble Recombinant Antigenic Domains of Toxoplasma gondii Secretory Organelles
Article Snippet: The pGEX-4T-1 vector and BL21 (DE3) pLysS E. coli were from GE Healthcare (Little Chalfont, UK). .. DH5α E. coli , PCR synthesis kit, T4 ligation kit and restriction enzyme digestion kit were from Enzynomics (Seoul, Korea).

Reverse Transcription Polymerase Chain Reaction:

Article Title: Thromboxane A2 modulates cisplatin-induced apoptosis through a Siva1-dependent mechanism
Article Snippet: The full-length open reading frame of Siva1 was cloned by RT-PCR using template RNA extracted from HeLa cells. .. Generation of the TP constructs in the pGEX-4T-1 vector (GE Healthcare) was described previously.

Generated:

Article Title: Thromboxane A2 modulates cisplatin-induced apoptosis through a Siva1-dependent mechanism
Article Snippet: The Cherry-HA-Siva1 construct was generated by inserting the HA-tagged Siva1 ORF in the pmCherry plasmid (cat. 632522, Clontech, Mountain View, CA, USA). .. Generation of the TP constructs in the pGEX-4T-1 vector (GE Healthcare) was described previously.

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).

DNA Sequencing:

Article Title: Profiling of Viral Proteins Expressed from the Genomic RNA of Japanese Encephalitis Virus Using a Panel of 15 Region-Specific Polyclonal Rabbit Antisera: Implications for Viral Gene Expression
Article Snippet: Next, the resulting PCR amplicons were individually ligated in-frame to the 3'-end of GST coding sequence in the pGex-4T-1 vector (GE Healthcare, Piscataway, NJ) using either the Eco RI and Xho I sites (for the first 14 PCR amplicons except NS5N-term and NS5C-term ) or the Bam HI and Xho I sites (for NS5N-term and NS5C-term ). .. The nucleotide sequences of the cloned cDNAs were verified by DNA sequencing.

Polymerase Chain Reaction:

Article Title: High Expression of Water-Soluble Recombinant Antigenic Domains of Toxoplasma gondii Secretory Organelles
Article Snippet: The pGEX-4T-1 vector and BL21 (DE3) pLysS E. coli were from GE Healthcare (Little Chalfont, UK). .. DH5α E. coli , PCR synthesis kit, T4 ligation kit and restriction enzyme digestion kit were from Enzynomics (Seoul, Korea).

Article Title: Profiling of Viral Proteins Expressed from the Genomic RNA of Japanese Encephalitis Virus Using a Panel of 15 Region-Specific Polyclonal Rabbit Antisera: Implications for Viral Gene Expression
Article Snippet: .. Next, the resulting PCR amplicons were individually ligated in-frame to the 3'-end of GST coding sequence in the pGex-4T-1 vector (GE Healthcare, Piscataway, NJ) using either the Eco RI and Xho I sites (for the first 14 PCR amplicons except NS5N-term and NS5C-term ) or the Bam HI and Xho I sites (for NS5N-term and NS5C-term ). .. The nucleotide sequences of the cloned cDNAs were verified by DNA sequencing.

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: The fused gene of BD2/3 was prepared by overlap extension PCR ( ). .. 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.

Article Title: Improved expression of recombinant fusion defensin gene plasmids packed with chitosan-derived nanoparticles and effect on antibacteria and mouse immunity
Article Snippet: The fused gene of BD2/3 was prepared by overlap extension PCR ( ). .. 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.

Article Title: High yield expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development
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). .. To generate the plasmid harboring the codon optimized nucleotide sequence, a codon optimized fragment encoding the first 107 amino acid residues of the N-terminus of the VP1 capsid protein was fused with the C-terminus of the VP1 protein using the 5' and 3' ends of above two DNA fragments and overlapping PCR [ ].

Article Title: Thromboxane A2 modulates cisplatin-induced apoptosis through a Siva1-dependent mechanism
Article Snippet: The PCR products were inserted into either the pcDNA3 expression vector (Life Technologies) to yield the Siva1-HA and Siva1ΔC-HA constructs or into the pRSETa expression vector (Invitrogen) to yield the 6 × His-Siva1-HA, 6 × His-Siva1ΔC-HA and 6 × His-Siva1ΔΔC-HA constructs. .. Generation of the TP constructs in the pGEX-4T-1 vector (GE Healthcare) was described previously.

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). ..

Affinity Purification:

Article Title: N-terminal Domain of the Spike Protein of Porcine Epidemic Diarrhea Virus as a New Candidate Molecule for a Mucosal Vaccine
Article Snippet: The amplified NTD231–501 gene was cloned into the pGEX 4T-1 expression vector (GE Healthcare Life Sciences, Buckinghamshire, UK) or the pET SUMO vector (Thermo Fisher Scientific, Waltham, VA, USA). .. The recombinant NTD231–501 protein expressed in the Escherichia coli (E. coli ) BL21 (DE3) strain was purified using a glutathione- or Ni-NTA-based affinity purification system (GE Healthcare Life Sciences and Thermo Fischer Scientific, respectively) according to the manufacturers' instructions.

Recombinant:

Article Title: N-terminal Domain of the Spike Protein of Porcine Epidemic Diarrhea Virus as a New Candidate Molecule for a Mucosal Vaccine
Article Snippet: Paragraph title: Construction of recombinant partial NTD protein of the PEDV S1 protein ... The amplified NTD231–501 gene was cloned into the pGEX 4T-1 expression vector (GE Healthcare Life Sciences, Buckinghamshire, UK) or the pET SUMO vector (Thermo Fisher Scientific, Waltham, VA, USA).

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.

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).

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: N-terminal Domain of the Spike Protein of Porcine Epidemic Diarrhea Virus as a New Candidate Molecule for a Mucosal Vaccine
Article Snippet: The amplified NTD231–501 gene was cloned into the pGEX 4T-1 expression vector (GE Healthcare Life Sciences, Buckinghamshire, UK) or the pET SUMO vector (Thermo Fisher Scientific, Waltham, VA, USA). .. The recombinant NTD231–501 protein expressed in the Escherichia coli (E. coli ) BL21 (DE3) strain was purified using a glutathione- or Ni-NTA-based affinity purification system (GE Healthcare Life Sciences and Thermo Fischer Scientific, respectively) according to the manufacturers' instructions.

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: 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).

Sequencing:

Article Title: Profiling of Viral Proteins Expressed from the Genomic RNA of Japanese Encephalitis Virus Using a Panel of 15 Region-Specific Polyclonal Rabbit Antisera: Implications for Viral Gene Expression
Article Snippet: .. Next, the resulting PCR amplicons were individually ligated in-frame to the 3'-end of GST coding sequence in the pGex-4T-1 vector (GE Healthcare, Piscataway, NJ) using either the Eco RI and Xho I sites (for the first 14 PCR amplicons except NS5N-term and NS5C-term ) or the Bam HI and Xho I sites (for NS5N-term and NS5C-term ). .. The nucleotide sequences of the cloned cDNAs were verified by DNA sequencing.

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: .. 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. .. Sequence of the resulting plasmid was confirmed by double restriction enzyme digestions and sequencing, and designated as pGB2B3.

Article Title: Improved expression of recombinant fusion defensin gene plasmids packed with chitosan-derived nanoparticles and effect on antibacteria and mouse immunity
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. .. Sequence of the resulting plasmid was confirmed by double restriction enzyme digestions and sequencing, and designated as pGB2B3.

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 expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development
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). .. To generate the plasmid harboring the codon optimized nucleotide sequence, a codon optimized fragment encoding the first 107 amino acid residues of the N-terminus of the VP1 capsid protein was fused with the C-terminus of the VP1 protein using the 5' and 3' ends of above two DNA fragments and overlapping PCR [ ].

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.

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.

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.

Plasmid Preparation:

Article Title: High Expression of Water-Soluble Recombinant Antigenic Domains of Toxoplasma gondii Secretory Organelles
Article Snippet: .. The pGEX-4T-1 vector and BL21 (DE3) pLysS E. coli were from GE Healthcare (Little Chalfont, UK). .. RNA extraction kit and plasmid prep kit were from Gene All (Seoul, Korea).

Article Title: Profiling of Viral Proteins Expressed from the Genomic RNA of Japanese Encephalitis Virus Using a Panel of 15 Region-Specific Polyclonal Rabbit Antisera: Implications for Viral Gene Expression
Article Snippet: .. Next, the resulting PCR amplicons were individually ligated in-frame to the 3'-end of GST coding sequence in the pGex-4T-1 vector (GE Healthcare, Piscataway, NJ) using either the Eco RI and Xho I sites (for the first 14 PCR amplicons except NS5N-term and NS5C-term ) or the Bam HI and Xho I sites (for NS5N-term and NS5C-term ). .. The nucleotide sequences of the cloned cDNAs were verified by DNA sequencing.

Article Title: N-terminal Domain of the Spike Protein of Porcine Epidemic Diarrhea Virus as a New Candidate Molecule for a Mucosal Vaccine
Article Snippet: .. The amplified NTD231–501 gene was cloned into the pGEX 4T-1 expression vector (GE Healthcare Life Sciences, Buckinghamshire, UK) or the pET SUMO vector (Thermo Fisher Scientific, Waltham, VA, USA). .. The recombinant NTD231–501 protein expressed in the Escherichia coli (E. coli ) BL21 (DE3) strain was purified using a glutathione- or Ni-NTA-based affinity purification system (GE Healthcare Life Sciences and Thermo Fischer Scientific, respectively) according to the manufacturers' instructions.

Article Title: Improved expression of recombinant fusion defensin gene plasmids packed with chitosan-derived nanoparticles and effect on antibacteria and mouse immunity
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. .. Sequence of the resulting plasmid was confirmed by double restriction enzyme digestions and sequencing, and designated as pGB2B3.

Article Title: Improved expression of recombinant fusion defensin gene plasmids packed with chitosan-derived nanoparticles and effect on antibacteria and mouse immunity
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. .. Sequence of the resulting plasmid was confirmed by double restriction enzyme digestions and sequencing, and designated as pGB2B3.

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 expression in a recombinant E. coli of a codon optimized chicken anemia virus capsid protein VP1 useful for vaccine development
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). .. This construct carries the VP1 gene flanked by the N-terminal glutathione-S-transferase (GST) tag and will express a GST-VP1 fusion protein.

Article Title: N-terminal Domain of the Spike Protein of Porcine Epidemic Diarrhea Virus as a New Candidate Molecule for a Mucosal Vaccine
Article Snippet: .. The NTD231–501 gene of the PEDV S1 protein was inserted into the pGEX 4T-1 expression vector containing a thrombin recognition site for cleaving the target protein. .. After expressing the construct in E. coli (BL21 Star (DE3)), the recombinant NTD231–501 protein yield was 20 mg/L.

Article Title: Thromboxane A2 modulates cisplatin-induced apoptosis through a Siva1-dependent mechanism
Article Snippet: .. Generation of the TP constructs in the pGEX-4T-1 vector (GE Healthcare) was described previously. .. Myc-Mdm2, Myc-TRAF2 and Myc-XIAP were all cloned by RT-PCR from HeLa cells mRNA and inserted into the pcDNA3 expression vector (Invitrogen).

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).

RNA Extraction:

Article Title: High Expression of Water-Soluble Recombinant Antigenic Domains of Toxoplasma gondii Secretory Organelles
Article Snippet: The pGEX-4T-1 vector and BL21 (DE3) pLysS E. coli were from GE Healthcare (Little Chalfont, UK). .. RNA extraction kit and plasmid prep kit were from Gene All (Seoul, Korea).

Positron Emission Tomography:

Article Title: N-terminal Domain of the Spike Protein of Porcine Epidemic Diarrhea Virus as a New Candidate Molecule for a Mucosal Vaccine
Article Snippet: .. The amplified NTD231–501 gene was cloned into the pGEX 4T-1 expression vector (GE Healthcare Life Sciences, Buckinghamshire, UK) or the pET SUMO vector (Thermo Fisher Scientific, Waltham, VA, USA). .. The recombinant NTD231–501 protein expressed in the Escherichia coli (E. coli ) BL21 (DE3) strain was purified using a glutathione- or Ni-NTA-based affinity purification system (GE Healthcare Life Sciences and Thermo Fischer Scientific, respectively) according to the manufacturers' instructions.

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).

CTG Assay:

Article Title: N-terminal Domain of the Spike Protein of Porcine Epidemic Diarrhea Virus as a New Candidate Molecule for a Mucosal Vaccine
Article Snippet: Construction of recombinant partial NTD protein of the PEDV S1 protein The partial NTD gene (encoding amino acids 231–501) was amplified from the S1 protein gene of the PEDV BM1 strain, which was synthesized by GeneScript (Piscataway, NJ, USA) using a specific primer set (F: 5′-CGC GGA TCC ACA GCT AAT TGC ATT-3′, R: 5′-CCG CTC GAG TCA AAA AGA AAT TGG CTG-3′). .. The amplified NTD231–501 gene was cloned into the pGEX 4T-1 expression vector (GE Healthcare Life Sciences, Buckinghamshire, UK) or the pET SUMO vector (Thermo Fisher Scientific, Waltham, VA, USA).

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.

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  • 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
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    Average 99 stars, based on 288 article reviews
    Price from $9.99 to $1999.99
    pgex 4t 1 - by Bioz Stars, 2020-02
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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 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    Average 91 stars, based on 6 article reviews
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    pgex 4t 1 expression plasmid - by Bioz Stars, 2020-02
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    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