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Stratagene bl21
Effect of fusion tags, E. coli strains and rare codon replacement on recombinant PiCV capsid protein expression. The three expression plasmids containing wild-type PiCV capsid gene were expressed in E. coli strains <t>BL21</t> (DE3), BL21 <t>(DE3)-pLysS</t> and BL21 (DE3)-CodonPlus-RIPL. Furthermore, similar plasmids but containing the codon-optimized PiCV capsid gene were also expressed in BL21 (DE3) and BL21 (DE3)-pLysS. All examined recombinant PiCV capsid proteins were analyzed by SDS-PAGE and Western-blotting. Anti-6xHis tag monoclonal antibody was used to recognize the 6xHis-tagged (A) and Trx-tagged (C) Cap proteins. Anti-GST monoclonal antibody was used for detecting GST-tagged (B) Cap proteins. Lane M, pre-stained protein marker; lane 1, 3, 5, 7 and 9, before IPTG induction; lane 2, 4, 6, 8 and 10, after IPTG induction for 4 hrs cultivation. The solid triangle pinpoints the expressed Cap protein.
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1) 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

Effect of fusion tags, E. coli strains and rare codon replacement on recombinant PiCV capsid protein expression. The three expression plasmids containing wild-type PiCV capsid gene were expressed in E. coli strains BL21 (DE3), BL21 (DE3)-pLysS and BL21 (DE3)-CodonPlus-RIPL. Furthermore, similar plasmids but containing the codon-optimized PiCV capsid gene were also expressed in BL21 (DE3) and BL21 (DE3)-pLysS. All examined recombinant PiCV capsid proteins were analyzed by SDS-PAGE and Western-blotting. Anti-6xHis tag monoclonal antibody was used to recognize the 6xHis-tagged (A) and Trx-tagged (C) Cap proteins. Anti-GST monoclonal antibody was used for detecting GST-tagged (B) Cap proteins. Lane M, pre-stained protein marker; lane 1, 3, 5, 7 and 9, before IPTG induction; lane 2, 4, 6, 8 and 10, after IPTG induction for 4 hrs cultivation. The solid triangle pinpoints the expressed Cap protein.
Figure Legend Snippet: Effect of fusion tags, E. coli strains and rare codon replacement on recombinant PiCV capsid protein expression. The three expression plasmids containing wild-type PiCV capsid gene were expressed in E. coli strains BL21 (DE3), BL21 (DE3)-pLysS and BL21 (DE3)-CodonPlus-RIPL. Furthermore, similar plasmids but containing the codon-optimized PiCV capsid gene were also expressed in BL21 (DE3) and BL21 (DE3)-pLysS. All examined recombinant PiCV capsid proteins were analyzed by SDS-PAGE and Western-blotting. Anti-6xHis tag monoclonal antibody was used to recognize the 6xHis-tagged (A) and Trx-tagged (C) Cap proteins. Anti-GST monoclonal antibody was used for detecting GST-tagged (B) Cap proteins. Lane M, pre-stained protein marker; lane 1, 3, 5, 7 and 9, before IPTG induction; lane 2, 4, 6, 8 and 10, after IPTG induction for 4 hrs cultivation. The solid triangle pinpoints the expressed Cap protein.

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

Growth kinetics and production profiles of recombinant PiCV capsid protein (Cap) in different E. coli strains. (A) The growth and protein production profiles of the three recombinant E. coli strains expressing either the GST-Cap or GST-Cap opt protein. Two strains, BL21 (DE3) and BL21 (DE3)-pLysS expressing GST-Cap opt , and one GST-Cap expressing strain BL21 (DE3)-CodonPlus-RIPL were used in this study post-induction. (B) Growth and protein production profiles of four recombinant E. coli strains expressing Trx-His-Cap or Trx-His-Cap opt protein. Two E. coli strains, BL21 (DE3) and BL21 (DE3)-pLysS were used to express Trx-His-Cap opt , and other two recombinant E. coli strains, BL21 (DE3)-CodonPlus-RIPL and BL21 (DE3)-pLysS were used to express Trx-His-Cap. Similar tests to those described above were used to detect the time course of protein expression after IPTG induction.
Figure Legend Snippet: Growth kinetics and production profiles of recombinant PiCV capsid protein (Cap) in different E. coli strains. (A) The growth and protein production profiles of the three recombinant E. coli strains expressing either the GST-Cap or GST-Cap opt protein. Two strains, BL21 (DE3) and BL21 (DE3)-pLysS expressing GST-Cap opt , and one GST-Cap expressing strain BL21 (DE3)-CodonPlus-RIPL were used in this study post-induction. (B) Growth and protein production profiles of four recombinant E. coli strains expressing Trx-His-Cap or Trx-His-Cap opt protein. Two E. coli strains, BL21 (DE3) and BL21 (DE3)-pLysS were used to express Trx-His-Cap opt , and other two recombinant E. coli strains, BL21 (DE3)-CodonPlus-RIPL and BL21 (DE3)-pLysS were used to express Trx-His-Cap. Similar tests to those described above were used to detect the time course of protein expression after IPTG induction.

Techniques Used: Recombinant, Expressing

The solubility of recombinant GST-Cap opt and Trx-His-Cap opt protein. The two proteins were expressed by E. coli strain BL21 (DE3)-pLysS transformed pGST-Cap opt and pTrx-His-Cap opt respectively. SDS-PAGE (A) was used to analyze the protein distribution pattern of suspension fraction and the pellet fraction. The soluble percent of two recombinant proteins was determined by measuring the intensity of target protein bands on coomassie blue-strained gels (B) . Lane M, pre-stained protein marker; lane I, total protein-expressed cell lysate; lane S, suspension fraction from centrifugal protein-expressed cell lysate; lane P, pellet fraction from centrifugal protein-expressed cell lysate. The solid triangle pinpoints the expressed Cap opt protein.
Figure Legend Snippet: The solubility of recombinant GST-Cap opt and Trx-His-Cap opt protein. The two proteins were expressed by E. coli strain BL21 (DE3)-pLysS transformed pGST-Cap opt and pTrx-His-Cap opt respectively. SDS-PAGE (A) was used to analyze the protein distribution pattern of suspension fraction and the pellet fraction. The soluble percent of two recombinant proteins was determined by measuring the intensity of target protein bands on coomassie blue-strained gels (B) . Lane M, pre-stained protein marker; lane I, total protein-expressed cell lysate; lane S, suspension fraction from centrifugal protein-expressed cell lysate; lane P, pellet fraction from centrifugal protein-expressed cell lysate. The solid triangle pinpoints the expressed Cap opt protein.

Techniques Used: Solubility, Recombinant, Transformation Assay, SDS Page, Staining, Marker

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

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

Journal: BMC Veterinary Research

doi: 10.1186/1746-6148-10-115

Effect of fusion tags, E. coli strains and rare codon replacement on recombinant PiCV capsid protein expression. The three expression plasmids containing wild-type PiCV capsid gene were expressed in E. coli strains BL21 (DE3), BL21 (DE3)-pLysS and BL21 (DE3)-CodonPlus-RIPL. Furthermore, similar plasmids but containing the codon-optimized PiCV capsid gene were also expressed in BL21 (DE3) and BL21 (DE3)-pLysS. All examined recombinant PiCV capsid proteins were analyzed by SDS-PAGE and Western-blotting. Anti-6xHis tag monoclonal antibody was used to recognize the 6xHis-tagged (A) and Trx-tagged (C) Cap proteins. Anti-GST monoclonal antibody was used for detecting GST-tagged (B) Cap proteins. Lane M, pre-stained protein marker; lane 1, 3, 5, 7 and 9, before IPTG induction; lane 2, 4, 6, 8 and 10, after IPTG induction for 4 hrs cultivation. The solid triangle pinpoints the expressed Cap protein.
Figure Legend Snippet: Effect of fusion tags, E. coli strains and rare codon replacement on recombinant PiCV capsid protein expression. The three expression plasmids containing wild-type PiCV capsid gene were expressed in E. coli strains BL21 (DE3), BL21 (DE3)-pLysS and BL21 (DE3)-CodonPlus-RIPL. Furthermore, similar plasmids but containing the codon-optimized PiCV capsid gene were also expressed in BL21 (DE3) and BL21 (DE3)-pLysS. All examined recombinant PiCV capsid proteins were analyzed by SDS-PAGE and Western-blotting. Anti-6xHis tag monoclonal antibody was used to recognize the 6xHis-tagged (A) and Trx-tagged (C) Cap proteins. Anti-GST monoclonal antibody was used for detecting GST-tagged (B) Cap proteins. Lane M, pre-stained protein marker; lane 1, 3, 5, 7 and 9, before IPTG induction; lane 2, 4, 6, 8 and 10, after IPTG induction for 4 hrs cultivation. The solid triangle pinpoints the expressed Cap protein.

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

Growth kinetics and production profiles of recombinant PiCV capsid protein (Cap) in different E. coli strains. (A) The growth and protein production profiles of the three recombinant E. coli strains expressing either the GST-Cap or GST-Cap opt protein. Two strains, BL21 (DE3) and BL21 (DE3)-pLysS expressing GST-Cap opt , and one GST-Cap expressing strain BL21 (DE3)-CodonPlus-RIPL were used in this study post-induction. (B) Growth and protein production profiles of four recombinant E. coli strains expressing Trx-His-Cap or Trx-His-Cap opt protein. Two E. coli strains, BL21 (DE3) and BL21 (DE3)-pLysS were used to express Trx-His-Cap opt , and other two recombinant E. coli strains, BL21 (DE3)-CodonPlus-RIPL and BL21 (DE3)-pLysS were used to express Trx-His-Cap. Similar tests to those described above were used to detect the time course of protein expression after IPTG induction.
Figure Legend Snippet: Growth kinetics and production profiles of recombinant PiCV capsid protein (Cap) in different E. coli strains. (A) The growth and protein production profiles of the three recombinant E. coli strains expressing either the GST-Cap or GST-Cap opt protein. Two strains, BL21 (DE3) and BL21 (DE3)-pLysS expressing GST-Cap opt , and one GST-Cap expressing strain BL21 (DE3)-CodonPlus-RIPL were used in this study post-induction. (B) Growth and protein production profiles of four recombinant E. coli strains expressing Trx-His-Cap or Trx-His-Cap opt protein. Two E. coli strains, BL21 (DE3) and BL21 (DE3)-pLysS were used to express Trx-His-Cap opt , and other two recombinant E. coli strains, BL21 (DE3)-CodonPlus-RIPL and BL21 (DE3)-pLysS were used to express Trx-His-Cap. Similar tests to those described above were used to detect the time course of protein expression after IPTG induction.

Techniques Used: Recombinant, Expressing

The solubility of recombinant GST-Cap opt and Trx-His-Cap opt protein. The two proteins were expressed by E. coli strain BL21 (DE3)-pLysS transformed pGST-Cap opt and pTrx-His-Cap opt respectively. SDS-PAGE (A) was used to analyze the protein distribution pattern of suspension fraction and the pellet fraction. The soluble percent of two recombinant proteins was determined by measuring the intensity of target protein bands on coomassie blue-strained gels (B) . Lane M, pre-stained protein marker; lane I, total protein-expressed cell lysate; lane S, suspension fraction from centrifugal protein-expressed cell lysate; lane P, pellet fraction from centrifugal protein-expressed cell lysate. The solid triangle pinpoints the expressed Cap opt protein.
Figure Legend Snippet: The solubility of recombinant GST-Cap opt and Trx-His-Cap opt protein. The two proteins were expressed by E. coli strain BL21 (DE3)-pLysS transformed pGST-Cap opt and pTrx-His-Cap opt respectively. SDS-PAGE (A) was used to analyze the protein distribution pattern of suspension fraction and the pellet fraction. The soluble percent of two recombinant proteins was determined by measuring the intensity of target protein bands on coomassie blue-strained gels (B) . Lane M, pre-stained protein marker; lane I, total protein-expressed cell lysate; lane S, suspension fraction from centrifugal protein-expressed cell lysate; lane P, pellet fraction from centrifugal protein-expressed cell lysate. The solid triangle pinpoints the expressed Cap opt protein.

Techniques Used: Solubility, Recombinant, Transformation Assay, SDS Page, Staining, Marker

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Protease Inhibitor:

Article Title: Drosophila Ten-m and Filamin Affect Motor Neuron Growth Cone Guidance
Article Snippet: The GST-Filamin fusion proteins, or GST alone, were expressed in BL21 (Stratagene) upon induction by isopropyl thiogalactoside (IPTG) and purified by incubating the resulting bacterial cell lysate with equilibrated Glutathione-sepharose beads (Pharmacia). .. The beads were washed five times with binding buffer (0.5% NP40 in PBS supplemented with EDTA free protease inhibitor cocktail from Roche).

Article Title: Differences in Erythrocyte Receptor Specificity of Different Parts of the Plasmodium falciparum Reticulocyte Binding Protein Homologue 2a ▿ Reticulocyte Binding Protein Homologue 2a ▿ †
Article Snippet: BL21(DE3) (Stratagene) was used to express rPfRH2a. .. Induced cultures were allowed to grow for 3 h at 37°C, resuspended in chilled lysis buffer (50 mM NaH2 PO4 [pH 8.0], 300 mM NaCl, 5 mM dithiothreitol [DTT]) with an EDTA-free protease inhibitor cocktail (Roche), and lysed by sonication.

Polymerase Chain Reaction:

Article Title: A Soluble Fucose-Specific Lectin from Aspergillus fumigatus Conidia - Structure, Specificity and Possible Role in Fungal Pathogenicity
Article Snippet: Amplified product was cloned into the expression vector pET29a (Novagen) using NdeI and HindIII for digestion of both PCR product and vector and T4 ligase for ligation, forming vector pET29-afl . .. Escherichia coli cells strain BL21(DE3)Gold (Stratagene) were transformed with the plasmid.

Article Title: Differences in Erythrocyte Receptor Specificity of Different Parts of the Plasmodium falciparum Reticulocyte Binding Protein Homologue 2a ▿ Reticulocyte Binding Protein Homologue 2a ▿ †
Article Snippet: The PCR products were digested with NdeI and XhoI and were cloned into expression vector pET24a(+) (Novagen) to generate a C-terminal His tag. .. BL21(DE3) (Stratagene) was used to express rPfRH2a.

Sonication:

Article Title: Monoclonal antibodies against all known variants of EspA: development of a simple diagnostic test for enteropathogenic Escherichia coli based on a key virulence factor
Article Snippet: Recombinant plasmids were transformed into BL21(D3) (Stratagene) by the calcium chloride method. .. After incubation for 30 min at 37 °C in the presence of lysozyme (10 mg ml−1 ) the cells were disrupted by sonication on ice.

Article Title: Differences in Erythrocyte Receptor Specificity of Different Parts of the Plasmodium falciparum Reticulocyte Binding Protein Homologue 2a ▿ Reticulocyte Binding Protein Homologue 2a ▿ †
Article Snippet: BL21(DE3) (Stratagene) was used to express rPfRH2a. .. Induced cultures were allowed to grow for 3 h at 37°C, resuspended in chilled lysis buffer (50 mM NaH2 PO4 [pH 8.0], 300 mM NaCl, 5 mM dithiothreitol [DTT]) with an EDTA-free protease inhibitor cocktail (Roche), and lysed by sonication.

Recombinant:

Article Title: Analyzing the Function of the Insert Region Found Between the α and β-Subunits in the Eukaryotic Nitrile Hydratase from Monosiga brevicollis
Article Snippet: .. The pSPTact plasmid was co-expressed with the previously reported pSMMαβ plasmid encoding recombinant MbNHase [ ] that had been freshly transformed into BL21(DE3) (Stratagene) competent cells. .. In addition, the pSPTact plasmid was co-expressed with the plasmids containing the Mb NHaseΔ238−257 and Mb NHaseΔ219−272 deletion mutant genes in BL21 magic cells.

Article Title: Monoclonal antibodies against all known variants of EspA: development of a simple diagnostic test for enteropathogenic Escherichia coli based on a key virulence factor
Article Snippet: .. Recombinant plasmids were transformed into BL21(D3) (Stratagene) by the calcium chloride method. .. Transformants were grown at 37 °C to mid-exponential phase in 400 ml Luria broth, induced by addition of IPTG to 1 mM and grown for a further 4 h. Recombinant proteins were isolated in the form of inclusion bodies.

Article Title: Differences in Erythrocyte Receptor Specificity of Different Parts of the Plasmodium falciparum Reticulocyte Binding Protein Homologue 2a ▿ Reticulocyte Binding Protein Homologue 2a ▿ †
Article Snippet: The recombinant PfRH2a protein (rPfRH2a) contains amino acids 2874 to 3052. .. BL21(DE3) (Stratagene) was used to express rPfRH2a.

Article Title: A Transient Kinetic Analysis of PRMT1 Catalysis
Article Snippet: Recombinant His-tagged rat PRMT1 was expressed in E. coli and purified with Ni-charged His6x-tag binding resin as reported previously ( ). .. Briefly, the PRMT1-pET28b plasmid was transformed into BL21(DE3) (Stratagene).

Ion Exchange Chromatography:

Article Title: Differences in Erythrocyte Receptor Specificity of Different Parts of the Plasmodium falciparum Reticulocyte Binding Protein Homologue 2a ▿ Reticulocyte Binding Protein Homologue 2a ▿ †
Article Snippet: BL21(DE3) (Stratagene) was used to express rPfRH2a. .. The recombinant protein was purified under native conditions using nickel-nitrilotriacetic acid (Ni-NTA) agarose (Qiagen), followed by ion-exchange chromatography using a Mono Q 5/50 GL column (Amersham) and gel filtration chromatography using a Superdex 75 column (Amersham) in phosphate-buffered saline (PBS), pH 7.4, with 180 mM NaCl.

Pull Down Assay:

Article Title: Drosophila Ten-m and Filamin Affect Motor Neuron Growth Cone Guidance
Article Snippet: Paragraph title: GST pull down assay ... The GST-Filamin fusion proteins, or GST alone, were expressed in BL21 (Stratagene) upon induction by isopropyl thiogalactoside (IPTG) and purified by incubating the resulting bacterial cell lysate with equilibrated Glutathione-sepharose beads (Pharmacia).

Mutagenesis:

Article Title: Analyzing the Function of the Insert Region Found Between the α and β-Subunits in the Eukaryotic Nitrile Hydratase from Monosiga brevicollis
Article Snippet: The pSPTact plasmid was co-expressed with the previously reported pSMMαβ plasmid encoding recombinant MbNHase [ ] that had been freshly transformed into BL21(DE3) (Stratagene) competent cells. .. In addition, the pSPTact plasmid was co-expressed with the plasmids containing the Mb NHaseΔ238−257 and Mb NHaseΔ219−272 deletion mutant genes in BL21 magic cells.

Article Title: Paramagnetism-Based NMR Restraints Lift Residual Dipolar Coupling Degeneracy in Multidomain Detergent-Solubilized Membrane Proteins
Article Snippet: Paragraph title: PLN expression, purification, and mutagenesis ... BL21(DE3) (Stratagene, San Diego, CA) E. coli cells were transformed with 100 ng of purified plasmid and selected on LB agar plates containing ampicillin (100 mg/mL).

Article Title: Improving the Resistance of a Eukaryotic ?-Barrel Protein to Thermal and Chemical Perturbation
Article Snippet: .. To produce these AfTom40 proteins, vectors containing wild-type and mutant AfTom40 were transformed into E.coli C41(DE3) (Lucigen) and BL21(DE3) (Stratagene, Agilent Technolgies) cells, respectively. .. Wild-type human Tom40A (hTom40AΔ1-82) and mutated human Tom40A (hTom40AΔ1-82mut ) were expressed forming inclusion bodies in E. coli BL21-CodonPlus (DE3) under tight control of expression from the T7 promoter.

Isolation:

Article Title: Monoclonal antibodies against all known variants of EspA: development of a simple diagnostic test for enteropathogenic Escherichia coli based on a key virulence factor
Article Snippet: Recombinant plasmids were transformed into BL21(D3) (Stratagene) by the calcium chloride method. .. Transformants were grown at 37 °C to mid-exponential phase in 400 ml Luria broth, induced by addition of IPTG to 1 mM and grown for a further 4 h. Recombinant proteins were isolated in the form of inclusion bodies.

Labeling:

Article Title: Drosophila Ten-m and Filamin Affect Motor Neuron Growth Cone Guidance
Article Snippet: The GST-Filamin fusion proteins, or GST alone, were expressed in BL21 (Stratagene) upon induction by isopropyl thiogalactoside (IPTG) and purified by incubating the resulting bacterial cell lysate with equilibrated Glutathione-sepharose beads (Pharmacia). .. Truncated Ten-m segments 1, 2, and 4 were in vitro translated and 35 S labeled with TNT T7 coupled reticulocyte lysate system (Promega).

Purification:

Article Title: Drosophila Ten-m and Filamin Affect Motor Neuron Growth Cone Guidance
Article Snippet: .. The GST-Filamin fusion proteins, or GST alone, were expressed in BL21 (Stratagene) upon induction by isopropyl thiogalactoside (IPTG) and purified by incubating the resulting bacterial cell lysate with equilibrated Glutathione-sepharose beads (Pharmacia). .. The beads were washed five times with binding buffer (0.5% NP40 in PBS supplemented with EDTA free protease inhibitor cocktail from Roche).

Article Title: Electron Spin Relaxation and Biochemical Characterization of the Hydrogenase Maturase HydF: Insights into [2Fe-2S] and [4Fe-4S] Cluster Communication and Hydrogenase Activation
Article Snippet: Paragraph title: Expression and Purification of HydF ... For expression of the HydFEG protein, all three of the constructs mentioned above were transformed into E. coli BL21(DE3)RIL, BL21(DE3)pLysS, or BL21(DE3) (Stratagene) cells, wherein all proteins are overexpressed in similar amounts.

Article Title: Analyzing the Function of the Insert Region Found Between the α and β-Subunits in the Eukaryotic Nitrile Hydratase from Monosiga brevicollis
Article Snippet: Paragraph title: Expression and Purification of WT MbNHase and the Mutants in the Absence or Presence of PtNHaseact . ... The pSPTact plasmid was co-expressed with the previously reported pSMMαβ plasmid encoding recombinant MbNHase [ ] that had been freshly transformed into BL21(DE3) (Stratagene) competent cells.

Article Title: Monoclonal antibodies against all known variants of EspA: development of a simple diagnostic test for enteropathogenic Escherichia coli based on a key virulence factor
Article Snippet: Paragraph title: Recombinant protein expression and purification. ... Recombinant plasmids were transformed into BL21(D3) (Stratagene) by the calcium chloride method.

Article Title: Paramagnetism-Based NMR Restraints Lift Residual Dipolar Coupling Degeneracy in Multidomain Detergent-Solubilized Membrane Proteins
Article Snippet: .. BL21(DE3) (Stratagene, San Diego, CA) E. coli cells were transformed with 100 ng of purified plasmid and selected on LB agar plates containing ampicillin (100 mg/mL). .. The L7C mutant was designed in a similar way.

Article Title: LAP degradation product reflects plasma kallikrein-dependent TGF-? activation in patients with hepatic fibrosis
Article Snippet: .. BL21 (Stratagene, La Jolla, CA) and purified using Glutathione Sepharose (GE Healthcare). .. Determination of the cleavage sites within LAP by PLK To identify the cleavage site in LAP during latent TGF-β1 activation by PLK, rhLAP β1 was incubated with PLK.

Article Title: Differences in Erythrocyte Receptor Specificity of Different Parts of the Plasmodium falciparum Reticulocyte Binding Protein Homologue 2a ▿ Reticulocyte Binding Protein Homologue 2a ▿ †
Article Snippet: Paragraph title: Cloning, expression, and purification of rPfRH2a in Escherichia coli. ... BL21(DE3) (Stratagene) was used to express rPfRH2a.

Article Title: A Transient Kinetic Analysis of PRMT1 Catalysis
Article Snippet: Paragraph title: Expression and purification of PRMT1 ... Briefly, the PRMT1-pET28b plasmid was transformed into BL21(DE3) (Stratagene).

Sequencing:

Article Title: A Soluble Fucose-Specific Lectin from Aspergillus fumigatus Conidia - Structure, Specificity and Possible Role in Fungal Pathogenicity
Article Snippet: Escherichia coli cells strain BL21(DE3)Gold (Stratagene) were transformed with the plasmid. .. The sequence of the plasmid pET29-afl and its presence in transformed E. coli cells were confirmed by restriction cleavage of re-isolated plasmid and its sequencing.

Article Title: Improving the Resistance of a Eukaryotic ?-Barrel Protein to Thermal and Chemical Perturbation
Article Snippet: The protein sequence of Tom40 from Aspergillus fumigatus was retrieved from the Aspergillus Genome Database. .. To produce these AfTom40 proteins, vectors containing wild-type and mutant AfTom40 were transformed into E.coli C41(DE3) (Lucigen) and BL21(DE3) (Stratagene, Agilent Technolgies) cells, respectively.

Article Title: Differences in Erythrocyte Receptor Specificity of Different Parts of the Plasmodium falciparum Reticulocyte Binding Protein Homologue 2a ▿ Reticulocyte Binding Protein Homologue 2a ▿ †
Article Snippet: PCR amplification was carried out using primers 5′-GACCATATGataaaaagtaaactagaatct-3′ and 5′-AACCTCGAGggtattatcatcagtagtact-3′ (lowercase letters indicate the PfRH2a gene sequence) from P. falciparum 3D7 genomic DNA (gDNA). .. BL21(DE3) (Stratagene) was used to express rPfRH2a.

Periodic Counter-current Chromatography:

Article Title: Comparative Genomics of NAD Biosynthesis in Cyanobacteria
Article Snippet: E. coli strains DH5α (Invitrogen, Carlsbad, CA), BL21, and BL21(DE3) (Stratagene, La Jolla, CA) were used for cloning and protein overexpression. .. Synechocystis sp. strain PCC 6803 strains were grown at 30°C in BG-11 medium ( ) under continuous white light (50 μmol photons m−2 s−1 ).

cDNA Library Assay:

Article Title: A Soluble Fucose-Specific Lectin from Aspergillus fumigatus Conidia - Structure, Specificity and Possible Role in Fungal Pathogenicity
Article Snippet: Expression system construction Commercially available cDNA library from A. fumigatus grown at 37°C (Stratagene) was used as a gene source. .. Escherichia coli cells strain BL21(DE3)Gold (Stratagene) were transformed with the plasmid.

SDS Page:

Article Title: Drosophila Ten-m and Filamin Affect Motor Neuron Growth Cone Guidance
Article Snippet: The GST-Filamin fusion proteins, or GST alone, were expressed in BL21 (Stratagene) upon induction by isopropyl thiogalactoside (IPTG) and purified by incubating the resulting bacterial cell lysate with equilibrated Glutathione-sepharose beads (Pharmacia). .. The fusion protein-bound beads and the 35 S labeled proteins were mixed, rotated for 2 hours at 4 degrees, washed five times with the binding buffer, then resolved on an SDS PAGE gel that was subsequently fixed, dried and exposed to X-ray film.

Plasmid Preparation:

Article Title: Drosophila Ten-m and Filamin Affect Motor Neuron Growth Cone Guidance
Article Snippet: GST pull down assay GST fusion protein constructs were made with the pGEX4T2 vector (Stratagene). .. The GST-Filamin fusion proteins, or GST alone, were expressed in BL21 (Stratagene) upon induction by isopropyl thiogalactoside (IPTG) and purified by incubating the resulting bacterial cell lysate with equilibrated Glutathione-sepharose beads (Pharmacia).

Article Title: Analyzing the Function of the Insert Region Found Between the α and β-Subunits in the Eukaryotic Nitrile Hydratase from Monosiga brevicollis
Article Snippet: .. The pSPTact plasmid was co-expressed with the previously reported pSMMαβ plasmid encoding recombinant MbNHase [ ] that had been freshly transformed into BL21(DE3) (Stratagene) competent cells. .. In addition, the pSPTact plasmid was co-expressed with the plasmids containing the Mb NHaseΔ238−257 and Mb NHaseΔ219−272 deletion mutant genes in BL21 magic cells.

Article Title: Paramagnetism-Based NMR Restraints Lift Residual Dipolar Coupling Degeneracy in Multidomain Detergent-Solubilized Membrane Proteins
Article Snippet: .. BL21(DE3) (Stratagene, San Diego, CA) E. coli cells were transformed with 100 ng of purified plasmid and selected on LB agar plates containing ampicillin (100 mg/mL). .. The L7C mutant was designed in a similar way.

Article Title: LAP degradation product reflects plasma kallikrein-dependent TGF-? activation in patients with hepatic fibrosis
Article Snippet: A plasmid encoding GST-rhLTGF-β1 was constructed by inserting a human TGF-β1 cDNA into the pGEX-6P-1 vector (GE Healthcare, Buckinghamshire, UK), and protein expressed in E. coli. .. BL21 (Stratagene, La Jolla, CA) and purified using Glutathione Sepharose (GE Healthcare).

Article Title: A Soluble Fucose-Specific Lectin from Aspergillus fumigatus Conidia - Structure, Specificity and Possible Role in Fungal Pathogenicity
Article Snippet: .. Escherichia coli cells strain BL21(DE3)Gold (Stratagene) were transformed with the plasmid. .. The sequence of the plasmid pET29-afl and its presence in transformed E. coli cells were confirmed by restriction cleavage of re-isolated plasmid and its sequencing.

Article Title: Improving the Resistance of a Eukaryotic ?-Barrel Protein to Thermal and Chemical Perturbation
Article Snippet: The corresponding gene and an AfTom40 gene coding for a protein with mutations K69H, N150H, S180L, S236A and K302H were synthesized and cloned into a pET24d vector (GeneArt / Invitrogen). .. To produce these AfTom40 proteins, vectors containing wild-type and mutant AfTom40 were transformed into E.coli C41(DE3) (Lucigen) and BL21(DE3) (Stratagene, Agilent Technolgies) cells, respectively.

Article Title: Differences in Erythrocyte Receptor Specificity of Different Parts of the Plasmodium falciparum Reticulocyte Binding Protein Homologue 2a ▿ Reticulocyte Binding Protein Homologue 2a ▿ †
Article Snippet: The PCR products were digested with NdeI and XhoI and were cloned into expression vector pET24a(+) (Novagen) to generate a C-terminal His tag. .. BL21(DE3) (Stratagene) was used to express rPfRH2a.

Article Title: A Transient Kinetic Analysis of PRMT1 Catalysis
Article Snippet: .. Briefly, the PRMT1-pET28b plasmid was transformed into BL21(DE3) (Stratagene). .. Transformed bacteria were incubated in LB media at 37°C for growth, and then 16°C for protein expression which was induced by 0.3 mM IPTG.

Binding Assay:

Article Title: Drosophila Ten-m and Filamin Affect Motor Neuron Growth Cone Guidance
Article Snippet: The GST-Filamin fusion proteins, or GST alone, were expressed in BL21 (Stratagene) upon induction by isopropyl thiogalactoside (IPTG) and purified by incubating the resulting bacterial cell lysate with equilibrated Glutathione-sepharose beads (Pharmacia). .. The beads were washed five times with binding buffer (0.5% NP40 in PBS supplemented with EDTA free protease inhibitor cocktail from Roche).

Article Title: A Transient Kinetic Analysis of PRMT1 Catalysis
Article Snippet: Recombinant His-tagged rat PRMT1 was expressed in E. coli and purified with Ni-charged His6x-tag binding resin as reported previously ( ). .. Briefly, the PRMT1-pET28b plasmid was transformed into BL21(DE3) (Stratagene).

In Vitro:

Article Title: Drosophila Ten-m and Filamin Affect Motor Neuron Growth Cone Guidance
Article Snippet: The GST-Filamin fusion proteins, or GST alone, were expressed in BL21 (Stratagene) upon induction by isopropyl thiogalactoside (IPTG) and purified by incubating the resulting bacterial cell lysate with equilibrated Glutathione-sepharose beads (Pharmacia). .. Truncated Ten-m segments 1, 2, and 4 were in vitro translated and 35 S labeled with TNT T7 coupled reticulocyte lysate system (Promega).

Article Title: A Role for E1B-AP5 in ATR Signaling Pathways during Adenovirus Infection
Article Snippet: Full-length E1B-AP5 cDNA ( ) was cloned into pGEX for expression in BL21 (Stratagene). .. Plasmids p11d-RPA70, p11d-RPA32, and p3d-RPA14, competent for in vitro transcription/translation, were described previously ( ).

Activation 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: Bacterial strains and cell inoculation Three commercial E. coli strains, BL21(DE3) (Invitrogen, Carlsbad, CA), BL21(DE3)CodonPlus-RIPL (Stratagene, La Jolla, CA) and BL21(DE3)pLysS (Stratagene, La Jolla, CA) were used and maintained at 37°C in the Luria-Bertani (LB) medium (1% tryptone, 0.5% yeast extract, 1% NaCl, pH 7.0). .. First, 0.5 mL of an overnight culture was inoculated into 50 mL LB medium to allow strain activation by growth at 37°C for around 3 hours, by which time the optical density of culture had reach 0.5 of OD600 .

Concentration Assay:

Article Title: Differences in Erythrocyte Receptor Specificity of Different Parts of the Plasmodium falciparum Reticulocyte Binding Protein Homologue 2a ▿ Reticulocyte Binding Protein Homologue 2a ▿ †
Article Snippet: BL21(DE3) (Stratagene) was used to express rPfRH2a. .. Isopropyl-β- d -thiogalactopyranoside (IPTG) at a final concentration of 1 mM was added to cultures at an A 600 of 0.6 to 0.8.

Lysis:

Article Title: Differences in Erythrocyte Receptor Specificity of Different Parts of the Plasmodium falciparum Reticulocyte Binding Protein Homologue 2a ▿ Reticulocyte Binding Protein Homologue 2a ▿ †
Article Snippet: BL21(DE3) (Stratagene) was used to express rPfRH2a. .. Induced cultures were allowed to grow for 3 h at 37°C, resuspended in chilled lysis buffer (50 mM NaH2 PO4 [pH 8.0], 300 mM NaCl, 5 mM dithiothreitol [DTT]) with an EDTA-free protease inhibitor cocktail (Roche), and lysed by sonication.

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    Stratagene bl21 codon plus de3 ril
    Western blot analysis of DsbA:HIV-1Pr expression on total cell extracts of <t>BL21-Codon</t> <t>Plus-(DE3)-RIL</t> E. coli cells containing the pET39-DsbA:HIV-1Pr plasmid and using different cultivation media. (Indicated below each panel): the cells were collected at different times after adding 1 mM IPTG (0, 0.5, 1, or 3 hours, as indicated above each lane). Protein expression induced at A) the early-exponential phase, B) the middle-exponential phase, and C) the stationary phase. An amount of cells corresponding to 1 mL (panel A) or 0.5 mL (panels B and C) of culture was loaded in each lane. D) Comparison of DsbA:HIV-1Pr production level obtained using different growth conditions. The first bar represents the best conditions identified using different E. coli strains (see Figure 1). In all cases variability among replicate cultures was lower than 10%. DsbA:HIV-1Pr expression was detected using anti-His-tag-specific antibodies. St: 0.5 μg of His-tagged recombinant D-amino acid oxidase.
    Bl21 Codon Plus De3 Ril, supplied by Stratagene, used in various techniques. Bioz Stars score: 86/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Stratagene ptd drbd expression utilized bl21 codon
    <t>PTD-DRBD</t> siRNA delivery into T cells and HUVECs ( a ) Flow cytometry analysis of dGFP RNAi knockdown decay kinetics of dividing Jurkat dGFP cells following treatment with GFP2 siRNA plus PTD-DRBD, Lipofection-2000 (Lipofection) or RNAiMAX (Lipofection 2), as indicated. ( b ) Quantitative RT-PCR analysis of endogenous GAPDH mRNA expression at 12 h post-treatment of GAPDH siRNA or GFP2 (Con) siRNA plus PTD-DRBD, GAPDH siRNA plus Lipofection-2000 (Lipofection) or RNAiMAX (Lipofection 2) in Jurkat cells, as indicated. Mean values normalized to β2 microglobulin and reported as percent of mock GAPDH control. ( c ) Flow cytometry histogram analysis of PTD-DRBD mediated CD4 or CD8 RNAi response at 1 day post-treatment of mouse primary T cells, as indicated. ( d ) Quantitative RT-PCR analysis of endogenous CD4, CD8 or CD90 mRNA expression at 12 and 24 h post-treatment of PTD-DRBD CD4 or CD8 siRNAs in primary T cells, as indicated. Mean values normalized to β2 microglobulin and reported as percent of mock control. *(P
    Ptd Drbd Expression Utilized Bl21 Codon, supplied by Stratagene, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    70
    Stratagene e coli protease deficient expression strain bl21
    Competitive binding of cannabinoid ligands on <t>BL21-107</t> membranes expressing CB2. The assay was performed in triplicate as described in Materials and Methods.
    E Coli Protease Deficient Expression Strain Bl21, supplied by Stratagene, used in various techniques. Bioz Stars score: 70/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Stratagene e coli bl21
    SDS-PAGE analysis of purified protein recombinant SllB in E. coli <t>BL21</t> cells. Lane M: Takara Protein Marker; lane 1, SDS-PAGE analysis of the recombinant S-layer protein before purification; lane 2, SDS-PAGE analysis of the purified recombinant protein.
    E Coli Bl21, supplied by Stratagene, used in various techniques. Bioz Stars score: 96/100, based on 133 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Western blot analysis of DsbA:HIV-1Pr expression on total cell extracts of BL21-Codon Plus-(DE3)-RIL E. coli cells containing the pET39-DsbA:HIV-1Pr plasmid and using different cultivation media. (Indicated below each panel): the cells were collected at different times after adding 1 mM IPTG (0, 0.5, 1, or 3 hours, as indicated above each lane). Protein expression induced at A) the early-exponential phase, B) the middle-exponential phase, and C) the stationary phase. An amount of cells corresponding to 1 mL (panel A) or 0.5 mL (panels B and C) of culture was loaded in each lane. D) Comparison of DsbA:HIV-1Pr production level obtained using different growth conditions. The first bar represents the best conditions identified using different E. coli strains (see Figure 1). In all cases variability among replicate cultures was lower than 10%. DsbA:HIV-1Pr expression was detected using anti-His-tag-specific antibodies. St: 0.5 μg of His-tagged recombinant D-amino acid oxidase.

    Journal: Microbial Cell Factories

    Article Title: Optimizing HIV-1 protease production in Escherichia coli as fusion protein

    doi: 10.1186/1475-2859-10-53

    Figure Lengend Snippet: Western blot analysis of DsbA:HIV-1Pr expression on total cell extracts of BL21-Codon Plus-(DE3)-RIL E. coli cells containing the pET39-DsbA:HIV-1Pr plasmid and using different cultivation media. (Indicated below each panel): the cells were collected at different times after adding 1 mM IPTG (0, 0.5, 1, or 3 hours, as indicated above each lane). Protein expression induced at A) the early-exponential phase, B) the middle-exponential phase, and C) the stationary phase. An amount of cells corresponding to 1 mL (panel A) or 0.5 mL (panels B and C) of culture was loaded in each lane. D) Comparison of DsbA:HIV-1Pr production level obtained using different growth conditions. The first bar represents the best conditions identified using different E. coli strains (see Figure 1). In all cases variability among replicate cultures was lower than 10%. DsbA:HIV-1Pr expression was detected using anti-His-tag-specific antibodies. St: 0.5 μg of His-tagged recombinant D-amino acid oxidase.

    Article Snippet: Strain, growth conditions, and enzyme expression For protein expression, plasmids containing the HIV-1Pr cDNA were transferred into different E. coli strains: BL21(DE3)pLysS (Novagen, Madison, Wisconsin, USA), BL21-Codon Plus(DE3)-RIL (Stratagene, La Jolla, California, USA), BL21-Star(DE3) (Invitrogen, Carslbad, California, USA), KRX (Promega, Madison, Wisconsin, USA), and C41(DE3), C41(DE3)pLysS, C43(DE3), C43(DE3)pLysS (Lucigen, Middleton, Wisconsin, USA) host cells.

    Techniques: Western Blot, Expressing, Plasmid Preparation, Recombinant

    Western blot analysis of DsbA:HIV-1Pr protein expression on total cell extracts of different E. coli strains containing the pET39-DsbA:HIV-1Pr plasmid. As detected using anti-His-tag-specific antibodies. Cells were grown in LB medium at 37°C, protein expression was induced using 1 mM IPTG at early-exponential phase, and cells were harvested after 1 hour. E. coli strains are: 1: BL21(DE3)pLysS; 2: C41(DE3); 3: C43(DE3); 4: C41(DE3)pLysS; 5: C43(DE3)pLysS; 6: KRX; 7: BL21-CodonPlus(DE3)-RIL; and 8: BL21(DE3)Star. In all cases variability among replicate cultures was lower than 10%. The amount of cells corresponding to 1 mL of culture was loaded in each lane. St: 0.5 μg of His-tagged recombinant D-amino acid oxidase.

    Journal: Microbial Cell Factories

    Article Title: Optimizing HIV-1 protease production in Escherichia coli as fusion protein

    doi: 10.1186/1475-2859-10-53

    Figure Lengend Snippet: Western blot analysis of DsbA:HIV-1Pr protein expression on total cell extracts of different E. coli strains containing the pET39-DsbA:HIV-1Pr plasmid. As detected using anti-His-tag-specific antibodies. Cells were grown in LB medium at 37°C, protein expression was induced using 1 mM IPTG at early-exponential phase, and cells were harvested after 1 hour. E. coli strains are: 1: BL21(DE3)pLysS; 2: C41(DE3); 3: C43(DE3); 4: C41(DE3)pLysS; 5: C43(DE3)pLysS; 6: KRX; 7: BL21-CodonPlus(DE3)-RIL; and 8: BL21(DE3)Star. In all cases variability among replicate cultures was lower than 10%. The amount of cells corresponding to 1 mL of culture was loaded in each lane. St: 0.5 μg of His-tagged recombinant D-amino acid oxidase.

    Article Snippet: Strain, growth conditions, and enzyme expression For protein expression, plasmids containing the HIV-1Pr cDNA were transferred into different E. coli strains: BL21(DE3)pLysS (Novagen, Madison, Wisconsin, USA), BL21-Codon Plus(DE3)-RIL (Stratagene, La Jolla, California, USA), BL21-Star(DE3) (Invitrogen, Carslbad, California, USA), KRX (Promega, Madison, Wisconsin, USA), and C41(DE3), C41(DE3)pLysS, C43(DE3), C43(DE3)pLysS (Lucigen, Middleton, Wisconsin, USA) host cells.

    Techniques: Western Blot, Expressing, Plasmid Preparation, Recombinant

    Amount of soluble DsbA:HIV-1Pr and phosphate salts effect on chimeric protein production . A) Comparison by means of Western blot analysis of the amount of DsbA:HIV-1Pr protein expression in the soluble (S) and insoluble (I) cell fractions as obtained after disruption of BL21(DE3)pLysS E. coli cells (growth in TB medium, protein expression induced at the early-exponential phase) or of BL21-Codon Plus-(DE3)-RIL E. coli cells (protein expression induced at the early and middle-exponential phase of growth). B) Western blot analysis of DsbA:HIV-1Pr protein expression in total cell extracts of recombinant BL21-Codon Plus-(DE3)-RIL E. coli cells grown in the presence of a phosphate salts buffer system. LB: cells grown in LB medium (control); a: cells grown in LB medium added of 6.78 g/L of NaH 2 PO 4 and 3 g/L of KH 2 PO 4 (such as in M9 broth); b: cells grown in LB medium to which 9.4 g/L of K 2 HPO 4 and 2.2 g/L of KH 2 PO 4 were added (such as in TB broth). Protein expression was induced in all samples with 1 mM IPTG; cells were harvested at 1 or 3 hours after adding IPTG. An amount of cells corresponding to 0.2 mL of culture was loaded in each lane. C) Summary results of DsbA:HIV-1Pr production (see panel B): the expression level of cells grown on LB medium is reported as 100% (= 1.8 mg/L). Cells were collected at 1 hour (empty bars) and 3 hours (black bars) from IPTG addition. Western blot analysis was carried out by using anti-His-tag-specific antibodies. St: 0.5 μg of His-tagged recombinant D-amino acid oxidase.

    Journal: Microbial Cell Factories

    Article Title: Optimizing HIV-1 protease production in Escherichia coli as fusion protein

    doi: 10.1186/1475-2859-10-53

    Figure Lengend Snippet: Amount of soluble DsbA:HIV-1Pr and phosphate salts effect on chimeric protein production . A) Comparison by means of Western blot analysis of the amount of DsbA:HIV-1Pr protein expression in the soluble (S) and insoluble (I) cell fractions as obtained after disruption of BL21(DE3)pLysS E. coli cells (growth in TB medium, protein expression induced at the early-exponential phase) or of BL21-Codon Plus-(DE3)-RIL E. coli cells (protein expression induced at the early and middle-exponential phase of growth). B) Western blot analysis of DsbA:HIV-1Pr protein expression in total cell extracts of recombinant BL21-Codon Plus-(DE3)-RIL E. coli cells grown in the presence of a phosphate salts buffer system. LB: cells grown in LB medium (control); a: cells grown in LB medium added of 6.78 g/L of NaH 2 PO 4 and 3 g/L of KH 2 PO 4 (such as in M9 broth); b: cells grown in LB medium to which 9.4 g/L of K 2 HPO 4 and 2.2 g/L of KH 2 PO 4 were added (such as in TB broth). Protein expression was induced in all samples with 1 mM IPTG; cells were harvested at 1 or 3 hours after adding IPTG. An amount of cells corresponding to 0.2 mL of culture was loaded in each lane. C) Summary results of DsbA:HIV-1Pr production (see panel B): the expression level of cells grown on LB medium is reported as 100% (= 1.8 mg/L). Cells were collected at 1 hour (empty bars) and 3 hours (black bars) from IPTG addition. Western blot analysis was carried out by using anti-His-tag-specific antibodies. St: 0.5 μg of His-tagged recombinant D-amino acid oxidase.

    Article Snippet: Strain, growth conditions, and enzyme expression For protein expression, plasmids containing the HIV-1Pr cDNA were transferred into different E. coli strains: BL21(DE3)pLysS (Novagen, Madison, Wisconsin, USA), BL21-Codon Plus(DE3)-RIL (Stratagene, La Jolla, California, USA), BL21-Star(DE3) (Invitrogen, Carslbad, California, USA), KRX (Promega, Madison, Wisconsin, USA), and C41(DE3), C41(DE3)pLysS, C43(DE3), C43(DE3)pLysS (Lucigen, Middleton, Wisconsin, USA) host cells.

    Techniques: Western Blot, Expressing, Recombinant

    Effect of temperature of growth after adding IPTG on the expression of DsbA:HIV-1Pr . Western blot analysis of soluble (S) and insoluble (I) cell extracts as obtained after disruption of recombinant BL21-Codon Plus-(DE3)-RIL E. coli cells expressing DsbA:HIV-1Pr fusion protein grown in TB (A) or M9 (B) media at different temperatures. Protein expression was induced by adding 1 mM IPTG at the early or middle-exponential phase of growth. Experimental conditions as in Figure 2. C) Summary results of soluble (empty bars) vs. insoluble (black bars) level of DsbA:HIV-1Pr expression in cells induced at the middle-exponential phase of growth and using TB or M9 media (see panels A and B).

    Journal: Microbial Cell Factories

    Article Title: Optimizing HIV-1 protease production in Escherichia coli as fusion protein

    doi: 10.1186/1475-2859-10-53

    Figure Lengend Snippet: Effect of temperature of growth after adding IPTG on the expression of DsbA:HIV-1Pr . Western blot analysis of soluble (S) and insoluble (I) cell extracts as obtained after disruption of recombinant BL21-Codon Plus-(DE3)-RIL E. coli cells expressing DsbA:HIV-1Pr fusion protein grown in TB (A) or M9 (B) media at different temperatures. Protein expression was induced by adding 1 mM IPTG at the early or middle-exponential phase of growth. Experimental conditions as in Figure 2. C) Summary results of soluble (empty bars) vs. insoluble (black bars) level of DsbA:HIV-1Pr expression in cells induced at the middle-exponential phase of growth and using TB or M9 media (see panels A and B).

    Article Snippet: Strain, growth conditions, and enzyme expression For protein expression, plasmids containing the HIV-1Pr cDNA were transferred into different E. coli strains: BL21(DE3)pLysS (Novagen, Madison, Wisconsin, USA), BL21-Codon Plus(DE3)-RIL (Stratagene, La Jolla, California, USA), BL21-Star(DE3) (Invitrogen, Carslbad, California, USA), KRX (Promega, Madison, Wisconsin, USA), and C41(DE3), C41(DE3)pLysS, C43(DE3), C43(DE3)pLysS (Lucigen, Middleton, Wisconsin, USA) host cells.

    Techniques: Expressing, Western Blot, Recombinant

    PTD-DRBD siRNA delivery into T cells and HUVECs ( a ) Flow cytometry analysis of dGFP RNAi knockdown decay kinetics of dividing Jurkat dGFP cells following treatment with GFP2 siRNA plus PTD-DRBD, Lipofection-2000 (Lipofection) or RNAiMAX (Lipofection 2), as indicated. ( b ) Quantitative RT-PCR analysis of endogenous GAPDH mRNA expression at 12 h post-treatment of GAPDH siRNA or GFP2 (Con) siRNA plus PTD-DRBD, GAPDH siRNA plus Lipofection-2000 (Lipofection) or RNAiMAX (Lipofection 2) in Jurkat cells, as indicated. Mean values normalized to β2 microglobulin and reported as percent of mock GAPDH control. ( c ) Flow cytometry histogram analysis of PTD-DRBD mediated CD4 or CD8 RNAi response at 1 day post-treatment of mouse primary T cells, as indicated. ( d ) Quantitative RT-PCR analysis of endogenous CD4, CD8 or CD90 mRNA expression at 12 and 24 h post-treatment of PTD-DRBD CD4 or CD8 siRNAs in primary T cells, as indicated. Mean values normalized to β2 microglobulin and reported as percent of mock control. *(P

    Journal: Nature biotechnology

    Article Title: Efficient siRNA Delivery into Primary Cells by Peptide Transduction-dsRNA Binding Domain (PTD-DRBD) Fusion Protein

    doi: 10.1038/nbt.1541

    Figure Lengend Snippet: PTD-DRBD siRNA delivery into T cells and HUVECs ( a ) Flow cytometry analysis of dGFP RNAi knockdown decay kinetics of dividing Jurkat dGFP cells following treatment with GFP2 siRNA plus PTD-DRBD, Lipofection-2000 (Lipofection) or RNAiMAX (Lipofection 2), as indicated. ( b ) Quantitative RT-PCR analysis of endogenous GAPDH mRNA expression at 12 h post-treatment of GAPDH siRNA or GFP2 (Con) siRNA plus PTD-DRBD, GAPDH siRNA plus Lipofection-2000 (Lipofection) or RNAiMAX (Lipofection 2) in Jurkat cells, as indicated. Mean values normalized to β2 microglobulin and reported as percent of mock GAPDH control. ( c ) Flow cytometry histogram analysis of PTD-DRBD mediated CD4 or CD8 RNAi response at 1 day post-treatment of mouse primary T cells, as indicated. ( d ) Quantitative RT-PCR analysis of endogenous CD4, CD8 or CD90 mRNA expression at 12 and 24 h post-treatment of PTD-DRBD CD4 or CD8 siRNAs in primary T cells, as indicated. Mean values normalized to β2 microglobulin and reported as percent of mock control. *(P

    Article Snippet: PTD-DRBD expression utilized BL21 codon plus (DH3) E.coli (Strategene) cells were transformed with pPTD-DRBD, cultured at 37°C in LB, then at 25°C for 12 h after induction with 400 μM IPTG.

    Techniques: Flow Cytometry, Cytometry, Quantitative RT-PCR, Expressing

    PTD-DRBD mediated siRNA delivery ( a ) Hypothetical cartoon of PTD-DRBD bound to siRNA. DRBD Ribbon structure derived from Ryter and Schultze 13 ( b ) Normalized RNAi knockdown of dGFP and dDsRed by PTD-DRBD:siRNA (left panel) and lipofection (right panel), as indicated, in H1299 dGFP/dDsRed cells. Mean values were normalized to percent control. ( c,d ) Single cell flow cytometry histogram analysis of dGFP RNAi response at 1 and 2 days post-treatment of H1299 dGFP/dDsRed cells, as indicated. ( e ) Flow cytometry analysis of dGFP RNAi knockdown decay kinetics following a single siRNA treatment of dividing H1299 dGFP/dDsRed cells. ( f ) Flow cytometry analysis of dGFP RNAi knockdown decay kinetics following multiple siRNA treatments of H1299 dGFP cells, as indicated. Mean values are normalized to percent control. ( g,h ) Quantitative RT-PCR analysis of endogenous GAPDH mRNA expression at 6 and 12 h post-treatment in H1299 cells, as indicated. Mean values normalized to β2 microglobulin and reported as percent of mock GAPDH control. **(P

    Journal: Nature biotechnology

    Article Title: Efficient siRNA Delivery into Primary Cells by Peptide Transduction-dsRNA Binding Domain (PTD-DRBD) Fusion Protein

    doi: 10.1038/nbt.1541

    Figure Lengend Snippet: PTD-DRBD mediated siRNA delivery ( a ) Hypothetical cartoon of PTD-DRBD bound to siRNA. DRBD Ribbon structure derived from Ryter and Schultze 13 ( b ) Normalized RNAi knockdown of dGFP and dDsRed by PTD-DRBD:siRNA (left panel) and lipofection (right panel), as indicated, in H1299 dGFP/dDsRed cells. Mean values were normalized to percent control. ( c,d ) Single cell flow cytometry histogram analysis of dGFP RNAi response at 1 and 2 days post-treatment of H1299 dGFP/dDsRed cells, as indicated. ( e ) Flow cytometry analysis of dGFP RNAi knockdown decay kinetics following a single siRNA treatment of dividing H1299 dGFP/dDsRed cells. ( f ) Flow cytometry analysis of dGFP RNAi knockdown decay kinetics following multiple siRNA treatments of H1299 dGFP cells, as indicated. Mean values are normalized to percent control. ( g,h ) Quantitative RT-PCR analysis of endogenous GAPDH mRNA expression at 6 and 12 h post-treatment in H1299 cells, as indicated. Mean values normalized to β2 microglobulin and reported as percent of mock GAPDH control. **(P

    Article Snippet: PTD-DRBD expression utilized BL21 codon plus (DH3) E.coli (Strategene) cells were transformed with pPTD-DRBD, cultured at 37°C in LB, then at 25°C for 12 h after induction with 400 μM IPTG.

    Techniques: Derivative Assay, Flow Cytometry, Cytometry, Quantitative RT-PCR, Expressing

    PTD-DRBD mediated RNAi responses ( a ) Fluorescent microscopy analysis of H9 hESCs constitutively expressing GFP treated with with PTD-DRBD delivered GFP2 siRNA at 2 days post-addition. Black line outlines hESC colony on mouse feeder cell background. ( b ) Oct4 immunoblot analysis in HUES9 hESCs treated with mock (PBS), PTD-DRBD delivered Oct4 or control siRNAs at 2 days post-addition. ( c ) Cell division curve of human HUES9 embryonic stem cells treated with mock (PBS), PTD-DRBD delivered Oct4 or control siRNAs, as indicated. ( d ) Immunohistochemistry analysis of Oct4 and SSEA4 expression in HUES9 hESCs at 2 days post-treatment with mock (PBS), PTD-DRB delivered Oct4 or control siRNAs. Anti-Oct4 antibodies (red), anti-SSEA-4 antibodies (green), genomic DNA (blue). ( e ) Immuno-histochemistry analysis of GATA6 and SSEA4 expression in HUES9 hESCs at 10 days post-treatment with mock (PBS), PTD-DRB delivered Oct4 or control siRNAs. Anti-GATA6 antibodies (red), anti-SSEA-4 antibodies (green), genomic DNA (blue). ( f, g ) Analysis of IFN-α and TNF-α induction in human PBMCs at 4 or 24 h post-treatment with mock (PBS), β-gal siRNA plus PTD-DRBD or plus Lipofection, as indicated. 10 μ/ml Imiquimod Imiquimod or 10 μg/ml LPS was used as a positive control for IFN-α or TNF-α, respectively. ( h ) Nasal and tracheal expressing ROSA26R-Luciferase transgenic mice were live animal imaged on day 0. Randomized groups of luciferase expressing mice were then treated with PBS, PTD-DRBD plus Luc siRNA or control GFP (Con) siRNA and monitored daily for luciferase expression, as indicated. Scale is in photons/s/cm 2 /sr. ( i ) Graph of percent Luciferase knockdown mice from (h) above. Luciferase expression was normalized to mock each day, error bar indicates s.e.m., n = 3 for each group with two luciferase readings performed per mouse per day.

    Journal: Nature biotechnology

    Article Title: Efficient siRNA Delivery into Primary Cells by Peptide Transduction-dsRNA Binding Domain (PTD-DRBD) Fusion Protein

    doi: 10.1038/nbt.1541

    Figure Lengend Snippet: PTD-DRBD mediated RNAi responses ( a ) Fluorescent microscopy analysis of H9 hESCs constitutively expressing GFP treated with with PTD-DRBD delivered GFP2 siRNA at 2 days post-addition. Black line outlines hESC colony on mouse feeder cell background. ( b ) Oct4 immunoblot analysis in HUES9 hESCs treated with mock (PBS), PTD-DRBD delivered Oct4 or control siRNAs at 2 days post-addition. ( c ) Cell division curve of human HUES9 embryonic stem cells treated with mock (PBS), PTD-DRBD delivered Oct4 or control siRNAs, as indicated. ( d ) Immunohistochemistry analysis of Oct4 and SSEA4 expression in HUES9 hESCs at 2 days post-treatment with mock (PBS), PTD-DRB delivered Oct4 or control siRNAs. Anti-Oct4 antibodies (red), anti-SSEA-4 antibodies (green), genomic DNA (blue). ( e ) Immuno-histochemistry analysis of GATA6 and SSEA4 expression in HUES9 hESCs at 10 days post-treatment with mock (PBS), PTD-DRB delivered Oct4 or control siRNAs. Anti-GATA6 antibodies (red), anti-SSEA-4 antibodies (green), genomic DNA (blue). ( f, g ) Analysis of IFN-α and TNF-α induction in human PBMCs at 4 or 24 h post-treatment with mock (PBS), β-gal siRNA plus PTD-DRBD or plus Lipofection, as indicated. 10 μ/ml Imiquimod Imiquimod or 10 μg/ml LPS was used as a positive control for IFN-α or TNF-α, respectively. ( h ) Nasal and tracheal expressing ROSA26R-Luciferase transgenic mice were live animal imaged on day 0. Randomized groups of luciferase expressing mice were then treated with PBS, PTD-DRBD plus Luc siRNA or control GFP (Con) siRNA and monitored daily for luciferase expression, as indicated. Scale is in photons/s/cm 2 /sr. ( i ) Graph of percent Luciferase knockdown mice from (h) above. Luciferase expression was normalized to mock each day, error bar indicates s.e.m., n = 3 for each group with two luciferase readings performed per mouse per day.

    Article Snippet: PTD-DRBD expression utilized BL21 codon plus (DH3) E.coli (Strategene) cells were transformed with pPTD-DRBD, cultured at 37°C in LB, then at 25°C for 12 h after induction with 400 μM IPTG.

    Techniques: Microscopy, Expressing, Immunohistochemistry, Positive Control, Luciferase, Transgenic Assay, Mouse Assay

    Competitive binding of cannabinoid ligands on BL21-107 membranes expressing CB2. The assay was performed in triplicate as described in Materials and Methods.

    Journal:

    Article Title: Expression of human peripheral cannabinoid receptor for structural studies

    doi: 10.1110/ps.051550305

    Figure Lengend Snippet: Competitive binding of cannabinoid ligands on BL21-107 membranes expressing CB2. The assay was performed in triplicate as described in Materials and Methods.

    Article Snippet: E. coli protease-deficient expression strain BL21 (DE3) was purchased from Stratagene.

    Techniques: Binding Assay, Expressing

    Ligand-binding assay on E. coli BL21-107 membranes

    Journal:

    Article Title: Expression of human peripheral cannabinoid receptor for structural studies

    doi: 10.1110/ps.051550305

    Figure Lengend Snippet: Ligand-binding assay on E. coli BL21-107 membranes

    Article Snippet: E. coli protease-deficient expression strain BL21 (DE3) was purchased from Stratagene.

    Techniques: Ligand Binding Assay

    Time course of CB2-107 expression in E. coli . ( A ) An overnight culture of BL21-107 cells was used to inoculate several incubation flasks containing equal volumes of media. Induction was started when cell density reached OD 600 = 0.4. Cell samples were

    Journal:

    Article Title: Expression of human peripheral cannabinoid receptor for structural studies

    doi: 10.1110/ps.051550305

    Figure Lengend Snippet: Time course of CB2-107 expression in E. coli . ( A ) An overnight culture of BL21-107 cells was used to inoculate several incubation flasks containing equal volumes of media. Induction was started when cell density reached OD 600 = 0.4. Cell samples were

    Article Snippet: E. coli protease-deficient expression strain BL21 (DE3) was purchased from Stratagene.

    Techniques: Expressing, Incubation

    Saturation binding of [ 3 H] CP-55,940 on BL21-107 membranes expressing CB2. The assay was performed in triplicate as described in Materials and Methods.

    Journal:

    Article Title: Expression of human peripheral cannabinoid receptor for structural studies

    doi: 10.1110/ps.051550305

    Figure Lengend Snippet: Saturation binding of [ 3 H] CP-55,940 on BL21-107 membranes expressing CB2. The assay was performed in triplicate as described in Materials and Methods.

    Article Snippet: E. coli protease-deficient expression strain BL21 (DE3) was purchased from Stratagene.

    Techniques: Binding Assay, Expressing

    SDS-PAGE analysis of purified protein recombinant SllB in E. coli BL21 cells. Lane M: Takara Protein Marker; lane 1, SDS-PAGE analysis of the recombinant S-layer protein before purification; lane 2, SDS-PAGE analysis of the purified recombinant protein.

    Journal: International Journal of Molecular Medicine

    Article Title: Cloning of the surface layer gene sllB from Bacillus sphaericus ATCC 14577 and its heterologous expression and purification

    doi: 10.3892/ijmm.2012.890

    Figure Lengend Snippet: SDS-PAGE analysis of purified protein recombinant SllB in E. coli BL21 cells. Lane M: Takara Protein Marker; lane 1, SDS-PAGE analysis of the recombinant S-layer protein before purification; lane 2, SDS-PAGE analysis of the purified recombinant protein.

    Article Snippet: 2.0 (Takara, no. DV801A), from which 0.5 μl was used to transform 100 μl of E. coli BL21 (DE3, Stratagene).

    Techniques: SDS Page, Purification, Recombinant, Marker

    A transmission electron microscopic observation on the E. coli BL21 with recombinant protein. (A) Normal E. coli BL21 was treated as control. (B) The E. coli BL21 cells recombinant S-layer protein. (C) Crystal lattice structures on surface of the E. coli BL21 cells recombinant S-layer protein.

    Journal: International Journal of Molecular Medicine

    Article Title: Cloning of the surface layer gene sllB from Bacillus sphaericus ATCC 14577 and its heterologous expression and purification

    doi: 10.3892/ijmm.2012.890

    Figure Lengend Snippet: A transmission electron microscopic observation on the E. coli BL21 with recombinant protein. (A) Normal E. coli BL21 was treated as control. (B) The E. coli BL21 cells recombinant S-layer protein. (C) Crystal lattice structures on surface of the E. coli BL21 cells recombinant S-layer protein.

    Article Snippet: 2.0 (Takara, no. DV801A), from which 0.5 μl was used to transform 100 μl of E. coli BL21 (DE3, Stratagene).

    Techniques: Transmission Assay, Recombinant

    Expression of sllB in E. coli BL21 cells. (A) SDS-PAGE analysis of rSllB protein; lane 1, the whole cell lysate of E. coli BL21 cells containing pET28a(+); lane 2, the supernatant of cells containing pET28a(+); lane 3, the pellet of cells containing pET28a(+); lane 4, the whole cell lysate of E. coli BL21 cells containing pET28a(+)- sllB ; lane 5, the supernatant of cell containing pET28a(+)- sllB ; lane 6, the pellet of cells containing pET28a(+)- sllB ; lane M represent Takara Protein Marker (Broad). (B) Western blot analysis of rSllB protein; lane 1, the whole cell lysate of E. coli BL21 cells containing pET28a(+); lane 2, the supernatant of cells containing pET28a(+); lane 3, the pellet of cells containing pET28a(+); lane 4, the whole cell lysate of E. coli BL21 cells containing pET28a(+)- sllB ; lane 5, the supernatant of cell containing pET28a(+)- sllB ; lane 6, the pellet of cells containing pET28a(+)- sllB ; M, Takara Protein Marker (Broad). Lane M1, Precision plus protein standards; lane M2, perfect protein marker. Note the band pointed with arrows is the recombinant S-layer protein.

    Journal: International Journal of Molecular Medicine

    Article Title: Cloning of the surface layer gene sllB from Bacillus sphaericus ATCC 14577 and its heterologous expression and purification

    doi: 10.3892/ijmm.2012.890

    Figure Lengend Snippet: Expression of sllB in E. coli BL21 cells. (A) SDS-PAGE analysis of rSllB protein; lane 1, the whole cell lysate of E. coli BL21 cells containing pET28a(+); lane 2, the supernatant of cells containing pET28a(+); lane 3, the pellet of cells containing pET28a(+); lane 4, the whole cell lysate of E. coli BL21 cells containing pET28a(+)- sllB ; lane 5, the supernatant of cell containing pET28a(+)- sllB ; lane 6, the pellet of cells containing pET28a(+)- sllB ; lane M represent Takara Protein Marker (Broad). (B) Western blot analysis of rSllB protein; lane 1, the whole cell lysate of E. coli BL21 cells containing pET28a(+); lane 2, the supernatant of cells containing pET28a(+); lane 3, the pellet of cells containing pET28a(+); lane 4, the whole cell lysate of E. coli BL21 cells containing pET28a(+)- sllB ; lane 5, the supernatant of cell containing pET28a(+)- sllB ; lane 6, the pellet of cells containing pET28a(+)- sllB ; M, Takara Protein Marker (Broad). Lane M1, Precision plus protein standards; lane M2, perfect protein marker. Note the band pointed with arrows is the recombinant S-layer protein.

    Article Snippet: 2.0 (Takara, no. DV801A), from which 0.5 μl was used to transform 100 μl of E. coli BL21 (DE3, Stratagene).

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