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SDS-PAGE (a) and western blot (b) analyses of the recombinant BirA protein expressed via pET24-BirA vector in <t>BL21</t> E. coli cells by addition of IPTG to the final concentration of 1 mM. (a) Lane 1: Protein molecular weight marker (Unstained protein molecular weight marker, Fermentas, Lithuania). Different induced colonies (lane 3–7) showed the same size of BirA (36.6 kDa) expression compared with un-induced sample (lane 2) (b) Western blot analysis of recombinant BirA protein by anti-His antibody specific for C-terminal polyhistidine tag indicated the specific 36.6 kDa protein band. Lane 1: Protein marker (PrestainedProtein Ladder, CinnaGen, Iran) Lane 2: Un-induced bacterial lysate. Lane 3: Recombinant BirA protein (36.6 kDa). Arrows indicate the target protein bands
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Article Title: Isolation, cloning, and expression of E. coli BirA gene for biotinylation applications

Journal: Advanced Biomedical Research

doi: 10.4103/2277-9175.161576

SDS-PAGE (a) and western blot (b) analyses of the recombinant BirA protein expressed via pET24-BirA vector in BL21 E. coli cells by addition of IPTG to the final concentration of 1 mM. (a) Lane 1: Protein molecular weight marker (Unstained protein molecular weight marker, Fermentas, Lithuania). Different induced colonies (lane 3–7) showed the same size of BirA (36.6 kDa) expression compared with un-induced sample (lane 2) (b) Western blot analysis of recombinant BirA protein by anti-His antibody specific for C-terminal polyhistidine tag indicated the specific 36.6 kDa protein band. Lane 1: Protein marker (PrestainedProtein Ladder, CinnaGen, Iran) Lane 2: Un-induced bacterial lysate. Lane 3: Recombinant BirA protein (36.6 kDa). Arrows indicate the target protein bands
Figure Legend Snippet: SDS-PAGE (a) and western blot (b) analyses of the recombinant BirA protein expressed via pET24-BirA vector in BL21 E. coli cells by addition of IPTG to the final concentration of 1 mM. (a) Lane 1: Protein molecular weight marker (Unstained protein molecular weight marker, Fermentas, Lithuania). Different induced colonies (lane 3–7) showed the same size of BirA (36.6 kDa) expression compared with un-induced sample (lane 2) (b) Western blot analysis of recombinant BirA protein by anti-His antibody specific for C-terminal polyhistidine tag indicated the specific 36.6 kDa protein band. Lane 1: Protein marker (PrestainedProtein Ladder, CinnaGen, Iran) Lane 2: Un-induced bacterial lysate. Lane 3: Recombinant BirA protein (36.6 kDa). Arrows indicate the target protein bands

Techniques Used: SDS Page, Western Blot, Recombinant, Plasmid Preparation, Concentration Assay, Molecular Weight, Marker, Expressing

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Flow Cytometry:

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Ligation:

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Cell Culture:

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Article Title:
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Liquid Chromatography:

Article Title: A Strategy for Synthesis of Pathogenic Human Immunoglobulin Free Light Chains in E. coli
Article Snippet: Plasmid amplification was carried out into the E. coli DH5α cells; insert sequence and reading frame were checked. .. E. coli BL21(DE3) cells (Merck Chemicals, Nottingham, UK) were transformed with the recombinant LC expression vectors and grown at 37°C in Luria-Bertani medium containing 100 µg/mL ampicillin. .. When the culture optical density at 600nm reached a value of 0.5–0.6, expression was induced by anhydrotetracycline addition to a final concentration of 0.43 µM.

Generated:

Article Title: A Sensitive Method for Detecting Zika Virus Antigen in Patients’ Whole-Blood Specimens as an Alternative Diagnostic Approach
Article Snippet: Antibodies were generated against the RNA helicase region of the NS3 protein of ZIKV (Asian genotype, isolate BeH819016, from Brazil). .. Expression was performed in E. coli BL21(DE3) cells (Merck Millipore), using an autoinduction protocol.

other:

Article Title: Recombinant expression and characterisation of the oxygen-sensitive 2-enoate reductase from Clostridium sporogenes
Article Snippet: C. sporogenes DSM 795 was cultivated in a medium containing cooked meat medium (5 g l−1 ; Lab M Limited), KH2 PO4 (5 g l−1 ), l -cysteine HCl (0.5 g l−1 ), and resazurin (0.001 % w/v), with the addition of agar (20 g l−1 ) when required [ ].

DNA Sequencing:

Article Title: Reduction of Spermidine Content Resulting from Inactivation of Two Arginine Decarboxylases Increases Biofilm Formation in Synechocystis sp. Strain PCC 6803
Article Snippet: The DNA sequences of slr1312 and slr0662 were verified by DNA sequencing. .. The E. coli BL21(DE3) strain (Merck Millipore, MA) containing the plasmids was grown in Luria-Bertani (LB) medium containing 100 μg/ml ampicillin overnight at 37°C.

Article Title: Use of Proteins Identified through a Functional Genomic Screen To Develop a Protein Subunit Vaccine That Provides Significant Protection against Virulent Streptococcus suis in Pigs
Article Snippet: The PCR products of candidate genes were cloned into the pET-30 Ek/LIC vector (Merck Millipore), and fusion plasmids were transformed into E. coli NovaBlue (Merck Millipore) according to the manufacturer's instructions. .. The positive recombinants were confirmed by PCR and DNA sequencing and then transformed into E. coli BL21(DE3) (Merck Millipore) for expression. .. Overnight cultures of E. coli BL21(DE3) strains carrying the recombinant plasmids were used to inoculate 1 to 6 liters fresh 2YT broth, grown to an OD595 of 0.6 at 37°C in broth supplemented with 100 μg/ml kanamycin, and then induced with 1 mM IPTG (isopropyl-β- d -1-thiogalactopyranoside; Sigma) at 37°C for 2, 4, and 24 h. Protein expression was checked by SDS-PAGE using whole-cell lysates.

Protein Concentration:

Article Title: A mutant β-glucosidase increases the rate of the cellulose enzymatic hydrolysis
Article Snippet: 2.2 Expression vectors pET46 coding for the wild-type and mutant Sfβgly were separately introduced in E. coli BL21(DE3) (Merck Millipore; Darmstad, Germany) by heat shock following manufacturer's procedures. .. The purity of the Sfβgly samples were checked by using SDS-PAGE .

Sequencing:

Article Title: The Cytolytic Activity of Vaginolysin Strictly Depends on Cholesterol and Is Potentiated by Human CD59
Article Snippet: The PCR fragment was sequenced and deduced amino acid sequence revealed 99% identity to ILY from S. intermedius ATCC 27335. .. PCR product comprising ILY coding gene lacking the putative signal sequence (aa residues 1–33) was cloned into pET28a(+) vector (Novagen/Merck KGaA, Darmstadt, Germany) and transformed into E. coli BL21(DE3) strain (Merck KGaA, Darmstadt, Germany). .. Recombinant N -terminally-hexahistidine-tagged ILY was purified from 2 L of culture using affinity chromatography on 6BCL-IDA Ni-sepharose (GE Healthcare, Helsinki, Finland) and ion-exchange chromatography on SP sepharose FF (GE Healthcare, Helsinki, Finland) according to manufacturer’s recommendations.

Article Title: A Sensitive Method for Detecting Zika Virus Antigen in Patients’ Whole-Blood Specimens as an Alternative Diagnostic Approach
Article Snippet: Sequence encoding for amino acid residues 1683–2123 of ZIKV BeH819016 polyprotein (GI:975885966) was codon optimized for Escherichia coli expression, obtained as synthetic DNA (Genscript) and cloned into pET28(a) expression vector. .. Expression was performed in E. coli BL21(DE3) cells (Merck Millipore), using an autoinduction protocol.

Article Title: β-III Tubulin Fragments Inhibit α-Synuclein Accumulation in Models of Multiple System Atrophy
Article Snippet: A peptide containing the putative α-syn-binding sequence (amino acids 235–282) as defined by GST pull-down assays and a control peptide (amino acids 283–331) were synthesized in a bacterial expression system. .. The resulting plasmids were transformed into Escherichia coli BL21(DE3)pLysS (Merck Millipore).

Sonication:

Article Title: Human Holliday junction resolvase GEN1 uses a chromodomain for efficient DNA recognition and cleavage
Article Snippet: All recombinant proteins were expressed in the E. coli BL21(DE3) pRIL strain (MerckMillipore, Darmstadt, Germany). .. All recombinant proteins were expressed in the E. coli BL21(DE3) pRIL strain (MerckMillipore, Darmstadt, Germany).

Affinity Purification:

Article Title: A Sensitive Method for Detecting Zika Virus Antigen in Patients’ Whole-Blood Specimens as an Alternative Diagnostic Approach
Article Snippet: Expression was performed in E. coli BL21(DE3) cells (Merck Millipore), using an autoinduction protocol. .. Recombinant soluble NS3 protein was purified to homogeneity by using Ni-affinity, ion-exchange, and size-exclusion chromatography as described previously [ ].

Article Title:
Article Snippet: Cells were cultured in DMEM containing 10% fetal bovine serum (Life Technologies) and full-length TFPI was purified from conditioned media by a two-step affinity purification protocol. .. The TFPI constructs TFPI-(1–150) and TFPI-(1–150)-Thr, containing a thrombin cleavage site between K1 and K2 were expressed as insoluble inclusion bodies in Escherichia coli BL21(DE3)pLysS (Merck, Darmstadt, Germany) using the expression vector pET19b (Merck).

Recombinant:

Article Title: The Cytolytic Activity of Vaginolysin Strictly Depends on Cholesterol and Is Potentiated by Human CD59
Article Snippet: PCR product comprising ILY coding gene lacking the putative signal sequence (aa residues 1–33) was cloned into pET28a(+) vector (Novagen/Merck KGaA, Darmstadt, Germany) and transformed into E. coli BL21(DE3) strain (Merck KGaA, Darmstadt, Germany). .. Recombinant N -terminally-hexahistidine-tagged ILY was purified from 2 L of culture using affinity chromatography on 6BCL-IDA Ni-sepharose (GE Healthcare, Helsinki, Finland) and ion-exchange chromatography on SP sepharose FF (GE Healthcare, Helsinki, Finland) according to manufacturer’s recommendations.

Article Title: Human Holliday junction resolvase GEN1 uses a chromodomain for efficient DNA recognition and cleavage
Article Snippet: Mutations were introduced by site-directed mutagenesis using Phusion Polymerase (NEB, Frankfurt/Main, Germany). .. All recombinant proteins were expressed in the E. coli BL21(DE3) pRIL strain (MerckMillipore, Darmstadt, Germany). .. Cells were grown at 37°C until mid-log phase and induced overnight with 0.2 mM IPTG at 16°C.

Article Title: A Strategy for Synthesis of Pathogenic Human Immunoglobulin Free Light Chains in E. coli
Article Snippet: Plasmid amplification was carried out into the E. coli DH5α cells; insert sequence and reading frame were checked. .. E. coli BL21(DE3) cells (Merck Chemicals, Nottingham, UK) were transformed with the recombinant LC expression vectors and grown at 37°C in Luria-Bertani medium containing 100 µg/mL ampicillin. .. When the culture optical density at 600nm reached a value of 0.5–0.6, expression was induced by anhydrotetracycline addition to a final concentration of 0.43 µM.

Article Title: Influence of N-glycosylation on effector functions and thermal stability of glycoengineered IgG1 monoclonal antibody with homogeneous glycoforms
Article Snippet: Paragraph title: Recombinant expression of GST-EndoS2 and GST-EndoS2-D184M ... Each plasmid was transformed into E. coli BL21(DE3) (Merck Millipore, Singles™ Competent Cells, Cat # 70235–3) and grown on an agar LB (Nacalai Tesque, Cat # 20066–95) plate containing 20 μg/mL kanamycin (Wako, Cat # 115–00342) overnight at 37 °C.

Article Title: A mutant β-glucosidase increases the rate of the cellulose enzymatic hydrolysis
Article Snippet: 2.2 Expression vectors pET46 coding for the wild-type and mutant Sfβgly were separately introduced in E. coli BL21(DE3) (Merck Millipore; Darmstad, Germany) by heat shock following manufacturer's procedures. .. Isolated colonies were used recombinantly express mutant and wild-type Sfβgly following the procedures previously described .

Article Title: Discovery of Sanggenon G as a natural cell-permeable small-molecular weight inhibitor of X-linked inhibitor of apoptosis protein (XIAP)
Article Snippet: Paragraph title: Production and purification of recombinant BIR3 protein ... Protein expression was performed in Escherichia coli BL21(DE3)pLysS (Merck Millipore, Germany).

Article Title: β-III Tubulin Fragments Inhibit α-Synuclein Accumulation in Models of Multiple System Atrophy
Article Snippet: The resulting plasmids were transformed into Escherichia coli BL21(DE3)pLysS (Merck Millipore). .. The resulting plasmids were transformed into Escherichia coli BL21(DE3)pLysS (Merck Millipore).

Molecular Weight:

Article Title:
Article Snippet: Homogeneous human full-length protein was characterized by mass spectrometry for molecular weight determination and SDS-PAGE at reducing and non-reducing conditions followed by Coomassie staining, silver staining, or immunoblotting. .. The TFPI constructs TFPI-(1–150) and TFPI-(1–150)-Thr, containing a thrombin cleavage site between K1 and K2 were expressed as insoluble inclusion bodies in Escherichia coli BL21(DE3)pLysS (Merck, Darmstadt, Germany) using the expression vector pET19b (Merck).

Mutagenesis:

Article Title: Human Holliday junction resolvase GEN1 uses a chromodomain for efficient DNA recognition and cleavage
Article Snippet: Mutations were introduced by site-directed mutagenesis using Phusion Polymerase (NEB, Frankfurt/Main, Germany). .. All recombinant proteins were expressed in the E. coli BL21(DE3) pRIL strain (MerckMillipore, Darmstadt, Germany).

Article Title: A mutant β-glucosidase increases the rate of the cellulose enzymatic hydrolysis
Article Snippet: These substrates were prepared in 100 mM sodium citrate - sodium phosphate buffer pH 6.0. .. 2.2 Expression vectors pET46 coding for the wild-type and mutant Sfβgly were separately introduced in E. coli BL21(DE3) (Merck Millipore; Darmstad, Germany) by heat shock following manufacturer's procedures. .. Isolated colonies were used recombinantly express mutant and wild-type Sfβgly following the procedures previously described .

Isolation:

Article Title: Isolation, cloning, and expression of E. coli BirA gene for biotinylation applications
Article Snippet: E. coli DH5α, a derivative of E. coli K-12,[ ] was used both as target strain for isolation of the BirA DNA fragment and also as the primary host for plasmid manipulations and cloning steps. pTG19-T cloning vector (Vivantis, Selangor DE, Malaysia) was used for TA cloning steps. .. E. coli BL21 (DE3) and pET-24a(+) plasmid (Merck KGaA, Darmstadt, Germany) were used as expression host and vector, respectively.

Article Title: Discovery of Sanggenon G as a natural cell-permeable small-molecular weight inhibitor of X-linked inhibitor of apoptosis protein (XIAP)
Article Snippet: Protein expression was performed in Escherichia coli BL21(DE3)pLysS (Merck Millipore, Germany). .. Protein expression was performed in Escherichia coli BL21(DE3)pLysS (Merck Millipore, Germany).

Purification:

Article Title: Direct induction of microtubule branching by microtubule nucleation factor SSNA1
Article Snippet: Paragraph title: Protein preparation and purification ... The proteins were expressed in E. coli BL21(DE3) (Merck, Darmstadt, Germany) by inducing with 0.4 mM IPTG (Carl Roth, Karlsruhe, Germany) for overnight at 18 °C.

Article Title: The Cytolytic Activity of Vaginolysin Strictly Depends on Cholesterol and Is Potentiated by Human CD59
Article Snippet: PCR product comprising ILY coding gene lacking the putative signal sequence (aa residues 1–33) was cloned into pET28a(+) vector (Novagen/Merck KGaA, Darmstadt, Germany) and transformed into E. coli BL21(DE3) strain (Merck KGaA, Darmstadt, Germany). .. Recombinant N -terminally-hexahistidine-tagged ILY was purified from 2 L of culture using affinity chromatography on 6BCL-IDA Ni-sepharose (GE Healthcare, Helsinki, Finland) and ion-exchange chromatography on SP sepharose FF (GE Healthcare, Helsinki, Finland) according to manufacturer’s recommendations.

Article Title: Reduction of Spermidine Content Resulting from Inactivation of Two Arginine Decarboxylases Increases Biofilm Formation in Synechocystis sp. Strain PCC 6803
Article Snippet: Paragraph title: Expression and purification of Adc1 and Adc2. ... The E. coli BL21(DE3) strain (Merck Millipore, MA) containing the plasmids was grown in Luria-Bertani (LB) medium containing 100 μg/ml ampicillin overnight at 37°C.

Article Title: Human Holliday junction resolvase GEN1 uses a chromodomain for efficient DNA recognition and cleavage
Article Snippet: Paragraph title: Protein expression and purification ... All recombinant proteins were expressed in the E. coli BL21(DE3) pRIL strain (MerckMillipore, Darmstadt, Germany).

Article Title: Common TFIIH recruitment mechanism in global genome and transcription-coupled repair subpathways
Article Snippet: Paragraph title: Protein expression and purification ... In brief, p62 or UVSSA was expressed as fusion product with glutathione S-transferase (GST) in a pGEX-4T vector (GE Healthcare) in Escherichia coli BL21(DE3) cells (Merck Millipore).

Article Title: A mutant β-glucosidase increases the rate of the cellulose enzymatic hydrolysis
Article Snippet: Paragraph title: Production and purification of the mutant and wild-type Sfβgly ... 2.2 Expression vectors pET46 coding for the wild-type and mutant Sfβgly were separately introduced in E. coli BL21(DE3) (Merck Millipore; Darmstad, Germany) by heat shock following manufacturer's procedures.

Article Title: UBE2B is implicated in myofibrillar protein loss in catabolic C2C12 myotubes) UBE2B is implicated in myofibrillar protein loss in catabolic C2C12 myotubes
Article Snippet: GST‐MuRF1 and UBE2B were expressed in Escherichia coli BL21(DE3) (Merck, Darmstadt, Germany) using 100 μM IPTG. .. GST‐MuRF1 and UBE2B were expressed in Escherichia coli BL21(DE3) (Merck, Darmstadt, Germany) using 100 μM IPTG.

Article Title: Discovery of Sanggenon G as a natural cell-permeable small-molecular weight inhibitor of X-linked inhibitor of apoptosis protein (XIAP)
Article Snippet: Paragraph title: Production and purification of recombinant BIR3 protein ... Protein expression was performed in Escherichia coli BL21(DE3)pLysS (Merck Millipore, Germany).

Article Title:
Article Snippet: Paragraph title: Expression, Purification, and Characterization of TFPI Proteins ... The TFPI constructs TFPI-(1–150) and TFPI-(1–150)-Thr, containing a thrombin cleavage site between K1 and K2 were expressed as insoluble inclusion bodies in Escherichia coli BL21(DE3)pLysS (Merck, Darmstadt, Germany) using the expression vector pET19b (Merck).

Article Title: A distal phenylalanine clamp in a hydrophobic channel controls the substrate specificity in the quorum-quenching metallo-γ-lactonase (AiiA) from Bacillus thuringiensis
Article Snippet: Paragraph title: Protein expression and purification ... Briefly, E. coli BL21(DE3) cells (Merck, Damstadt, Germany) transformed with the pMAL-t-AiiA vector were grown and induced with 0.5 mM IPTG.

Article Title: β-III Tubulin Fragments Inhibit α-Synuclein Accumulation in Models of Multiple System Atrophy
Article Snippet: The resulting plasmids were transformed into Escherichia coli BL21(DE3)pLysS (Merck Millipore). .. The resulting plasmids were transformed into Escherichia coli BL21(DE3)pLysS (Merck Millipore).

Article Title:
Article Snippet: Paragraph title: Expression, Purification, and Renaturation of CV-1 to CV-5, CLC, IC-7, IL-6, and CNTF ... Expression of CV-1 to CV-5, CLC, IC-7, IL-6, and CNTF was performed in Escherichia coli BL21(DE3) (Merck KGaA).

Polymerase Chain Reaction:

Article Title: The Cytolytic Activity of Vaginolysin Strictly Depends on Cholesterol and Is Potentiated by Human CD59
Article Snippet: The PCR fragment was sequenced and deduced amino acid sequence revealed 99% identity to ILY from S. intermedius ATCC 27335. .. PCR product comprising ILY coding gene lacking the putative signal sequence (aa residues 1–33) was cloned into pET28a(+) vector (Novagen/Merck KGaA, Darmstadt, Germany) and transformed into E. coli BL21(DE3) strain (Merck KGaA, Darmstadt, Germany). .. Recombinant N -terminally-hexahistidine-tagged ILY was purified from 2 L of culture using affinity chromatography on 6BCL-IDA Ni-sepharose (GE Healthcare, Helsinki, Finland) and ion-exchange chromatography on SP sepharose FF (GE Healthcare, Helsinki, Finland) according to manufacturer’s recommendations.

Article Title: Reduction of Spermidine Content Resulting from Inactivation of Two Arginine Decarboxylases Increases Biofilm Formation in Synechocystis sp. Strain PCC 6803
Article Snippet: The PCR products were digested with NdeI and BamHI and ligated into the corresponding sites of plasmid pET16b (Merck Millipore, MA). .. The E. coli BL21(DE3) strain (Merck Millipore, MA) containing the plasmids was grown in Luria-Bertani (LB) medium containing 100 μg/ml ampicillin overnight at 37°C.

Article Title: Human Holliday junction resolvase GEN1 uses a chromodomain for efficient DNA recognition and cleavage
Article Snippet: Wild type human GEN1 and truncations thereof (residues 2-551, 2-505, 2-464, 2-389) were amplified by PCR from IMAGE clone 40125755 (Mammalian Gene collection, natural variant S92T, S310N, UniProtID Q17RS7) and cloned into a self-made ligation-independent cloning vector with various C-terminal tags followed by His8. .. All recombinant proteins were expressed in the E. coli BL21(DE3) pRIL strain (MerckMillipore, Darmstadt, Germany).

Article Title: Use of Proteins Identified through a Functional Genomic Screen To Develop a Protein Subunit Vaccine That Provides Significant Protection against Virulent Streptococcus suis in Pigs
Article Snippet: The PCR products of candidate genes were cloned into the pET-30 Ek/LIC vector (Merck Millipore), and fusion plasmids were transformed into E. coli NovaBlue (Merck Millipore) according to the manufacturer's instructions. .. The positive recombinants were confirmed by PCR and DNA sequencing and then transformed into E. coli BL21(DE3) (Merck Millipore) for expression. .. Overnight cultures of E. coli BL21(DE3) strains carrying the recombinant plasmids were used to inoculate 1 to 6 liters fresh 2YT broth, grown to an OD595 of 0.6 at 37°C in broth supplemented with 100 μg/ml kanamycin, and then induced with 1 mM IPTG (isopropyl-β- d -1-thiogalactopyranoside; Sigma) at 37°C for 2, 4, and 24 h. Protein expression was checked by SDS-PAGE using whole-cell lysates.

Article Title: Nectrisine Biosynthesis Genes in Thelonectria discophora SANK 18292: Identification and Functional Analysis
Article Snippet: Paragraph title: Bacterial strains, nucleic acid manipulations, and PCR primers. ... E. coli BL21(DE3) (Merck Millipore, Billerica, MA) was used for protein production.

Article Title: Discovery of Sanggenon G as a natural cell-permeable small-molecular weight inhibitor of X-linked inhibitor of apoptosis protein (XIAP)
Article Snippet: 2.8 DNA encoding BIR3 of human XIAP protein was amplified by PCR and cloned into the pET30a(+) vector (Merck Millipore, Germany) via the NdeI and SalI restriction sites. .. Protein expression was performed in Escherichia coli BL21(DE3)pLysS (Merck Millipore, Germany).

Article Title: β-III Tubulin Fragments Inhibit α-Synuclein Accumulation in Models of Multiple System Atrophy
Article Snippet: Partial cDNA fragments encoding these sequences were amplified, and a BamHI site was added at the 5′ end and a stop codon to the 3′ end by PCR ( ). .. The resulting plasmids were transformed into Escherichia coli BL21(DE3)pLysS (Merck Millipore).

Positron Emission Tomography:

Article Title: Isolation, cloning, and expression of E. coli BirA gene for biotinylation applications
Article Snippet: E. coli DH5α, a derivative of E. coli K-12,[ ] was used both as target strain for isolation of the BirA DNA fragment and also as the primary host for plasmid manipulations and cloning steps. pTG19-T cloning vector (Vivantis, Selangor DE, Malaysia) was used for TA cloning steps. .. E. coli BL21 (DE3) and pET-24a(+) plasmid (Merck KGaA, Darmstadt, Germany) were used as expression host and vector, respectively. .. Bacterial strains were cultured in Luria-Bertani medium (supplemented with 50 μg/mL kanamycin when required).

Article Title: Use of Proteins Identified through a Functional Genomic Screen To Develop a Protein Subunit Vaccine That Provides Significant Protection against Virulent Streptococcus suis in Pigs
Article Snippet: The PCR products of candidate genes were cloned into the pET-30 Ek/LIC vector (Merck Millipore), and fusion plasmids were transformed into E. coli NovaBlue (Merck Millipore) according to the manufacturer's instructions. .. The positive recombinants were confirmed by PCR and DNA sequencing and then transformed into E. coli BL21(DE3) (Merck Millipore) for expression.

Affinity Column:

Article Title:
Article Snippet: The TFPI constructs TFPI-(1–150) and TFPI-(1–150)-Thr, containing a thrombin cleavage site between K1 and K2 were expressed as insoluble inclusion bodies in Escherichia coli BL21(DE3)pLysS (Merck, Darmstadt, Germany) using the expression vector pET19b (Merck). .. The TFPI constructs TFPI-(1–150) and TFPI-(1–150)-Thr, containing a thrombin cleavage site between K1 and K2 were expressed as insoluble inclusion bodies in Escherichia coli BL21(DE3)pLysS (Merck, Darmstadt, Germany) using the expression vector pET19b (Merck).

Periodic Counter-current Chromatography:

Article Title: Reduction of Spermidine Content Resulting from Inactivation of Two Arginine Decarboxylases Increases Biofilm Formation in Synechocystis sp. Strain PCC 6803
Article Snippet: The genes encoding arginine decarboxylases ( slr1312 and slr0662 ) were amplified by PCR from Synechocystis sp. strain PCC 6803 genomic DNA as the template with a set of primers for adc1 ( slr1312 ) (5′-GGAATTCCATATGGGGGAAGAACCTGTGCCGGC-3′ and 5′-GGAGGATCCTCAACTTAGATAGGTGTAGCGCCG-3′) or adc2 ( slr0662 ) (5′-GGAATTCCATATGGAAGGGCAGTCAATCGAACTAG-3′ and 5′-GGAGGATCCTTAGCTGGTGTGGATGCCTGAAGTGG-3′). .. The E. coli BL21(DE3) strain (Merck Millipore, MA) containing the plasmids was grown in Luria-Bertani (LB) medium containing 100 μg/ml ampicillin overnight at 37°C.

Lysis:

Article Title: Reduction of Spermidine Content Resulting from Inactivation of Two Arginine Decarboxylases Increases Biofilm Formation in Synechocystis sp. Strain PCC 6803
Article Snippet: The E. coli BL21(DE3) strain (Merck Millipore, MA) containing the plasmids was grown in Luria-Bertani (LB) medium containing 100 μg/ml ampicillin overnight at 37°C. .. When the culture reached an OD600 of 0.6, isopropyl-β- d -thiogalactopyranoside (IPTG) was added to a final concentration of 0.4 mM and the culture was grown at 20°C for another 20 h. The cells were harvested by centrifugation (5,200 × g ) for 15 min.

Article Title: Human Holliday junction resolvase GEN1 uses a chromodomain for efficient DNA recognition and cleavage
Article Snippet: All recombinant proteins were expressed in the E. coli BL21(DE3) pRIL strain (MerckMillipore, Darmstadt, Germany). .. All recombinant proteins were expressed in the E. coli BL21(DE3) pRIL strain (MerckMillipore, Darmstadt, Germany).

Article Title: Discovery of Sanggenon G as a natural cell-permeable small-molecular weight inhibitor of X-linked inhibitor of apoptosis protein (XIAP)
Article Snippet: Protein expression was performed in Escherichia coli BL21(DE3)pLysS (Merck Millipore, Germany). .. Protein expression was performed in Escherichia coli BL21(DE3)pLysS (Merck Millipore, Germany).

Silver Staining:

Article Title:
Article Snippet: Homogeneous human full-length protein was characterized by mass spectrometry for molecular weight determination and SDS-PAGE at reducing and non-reducing conditions followed by Coomassie staining, silver staining, or immunoblotting. .. The TFPI constructs TFPI-(1–150) and TFPI-(1–150)-Thr, containing a thrombin cleavage site between K1 and K2 were expressed as insoluble inclusion bodies in Escherichia coli BL21(DE3)pLysS (Merck, Darmstadt, Germany) using the expression vector pET19b (Merck).

SDS Page:

Article Title: A mutant β-glucosidase increases the rate of the cellulose enzymatic hydrolysis
Article Snippet: 2.2 Expression vectors pET46 coding for the wild-type and mutant Sfβgly were separately introduced in E. coli BL21(DE3) (Merck Millipore; Darmstad, Germany) by heat shock following manufacturer's procedures. .. 2.2 Expression vectors pET46 coding for the wild-type and mutant Sfβgly were separately introduced in E. coli BL21(DE3) (Merck Millipore; Darmstad, Germany) by heat shock following manufacturer's procedures.

Article Title:
Article Snippet: Homogeneous human full-length protein was characterized by mass spectrometry for molecular weight determination and SDS-PAGE at reducing and non-reducing conditions followed by Coomassie staining, silver staining, or immunoblotting. .. The TFPI constructs TFPI-(1–150) and TFPI-(1–150)-Thr, containing a thrombin cleavage site between K1 and K2 were expressed as insoluble inclusion bodies in Escherichia coli BL21(DE3)pLysS (Merck, Darmstadt, Germany) using the expression vector pET19b (Merck).

Plasmid Preparation:

Article Title: Isolation, cloning, and expression of E. coli BirA gene for biotinylation applications
Article Snippet: E. coli DH5α, a derivative of E. coli K-12,[ ] was used both as target strain for isolation of the BirA DNA fragment and also as the primary host for plasmid manipulations and cloning steps. pTG19-T cloning vector (Vivantis, Selangor DE, Malaysia) was used for TA cloning steps. .. E. coli BL21 (DE3) and pET-24a(+) plasmid (Merck KGaA, Darmstadt, Germany) were used as expression host and vector, respectively. .. Bacterial strains were cultured in Luria-Bertani medium (supplemented with 50 μg/mL kanamycin when required).

Article Title: The Cytolytic Activity of Vaginolysin Strictly Depends on Cholesterol and Is Potentiated by Human CD59
Article Snippet: The PCR fragment was sequenced and deduced amino acid sequence revealed 99% identity to ILY from S. intermedius ATCC 27335. .. PCR product comprising ILY coding gene lacking the putative signal sequence (aa residues 1–33) was cloned into pET28a(+) vector (Novagen/Merck KGaA, Darmstadt, Germany) and transformed into E. coli BL21(DE3) strain (Merck KGaA, Darmstadt, Germany). .. Recombinant N -terminally-hexahistidine-tagged ILY was purified from 2 L of culture using affinity chromatography on 6BCL-IDA Ni-sepharose (GE Healthcare, Helsinki, Finland) and ion-exchange chromatography on SP sepharose FF (GE Healthcare, Helsinki, Finland) according to manufacturer’s recommendations.

Article Title: Reduction of Spermidine Content Resulting from Inactivation of Two Arginine Decarboxylases Increases Biofilm Formation in Synechocystis sp. Strain PCC 6803
Article Snippet: The PCR products were digested with NdeI and BamHI and ligated into the corresponding sites of plasmid pET16b (Merck Millipore, MA). .. The E. coli BL21(DE3) strain (Merck Millipore, MA) containing the plasmids was grown in Luria-Bertani (LB) medium containing 100 μg/ml ampicillin overnight at 37°C.

Article Title: Human Holliday junction resolvase GEN1 uses a chromodomain for efficient DNA recognition and cleavage
Article Snippet: Wild type human GEN1 and truncations thereof (residues 2-551, 2-505, 2-464, 2-389) were amplified by PCR from IMAGE clone 40125755 (Mammalian Gene collection, natural variant S92T, S310N, UniProtID Q17RS7) and cloned into a self-made ligation-independent cloning vector with various C-terminal tags followed by His8. .. All recombinant proteins were expressed in the E. coli BL21(DE3) pRIL strain (MerckMillipore, Darmstadt, Germany).

Article Title: Influence of N-glycosylation on effector functions and thermal stability of glycoengineered IgG1 monoclonal antibody with homogeneous glycoforms
Article Snippet: The replacement of GAT (D: Asp) nucleic acid sequence (ndoS2 gene fragment from GenBank accession number KC155346, from 632 to 634) with ATG (M: Met) was incorporated, resulting in corresponding D184M mutation. pET-42_a_ndoS2-D184M plasmid was then obtained with the same procedure as the wild-type plasmid. .. Each plasmid was transformed into E. coli BL21(DE3) (Merck Millipore, Singles™ Competent Cells, Cat # 70235–3) and grown on an agar LB (Nacalai Tesque, Cat # 20066–95) plate containing 20 μg/mL kanamycin (Wako, Cat # 115–00342) overnight at 37 °C. .. Each recombinant clone was selected and cultured in a liquid LB medium containing 20 μg/mL kanamycin for six hours at 30 °C to obtain research cell banks (RCB), maintained as stocks in glycerol (Nacalai Tesque, Cat # 17018–83).

Article Title: Use of Proteins Identified through a Functional Genomic Screen To Develop a Protein Subunit Vaccine That Provides Significant Protection against Virulent Streptococcus suis in Pigs
Article Snippet: The PCR products of candidate genes were cloned into the pET-30 Ek/LIC vector (Merck Millipore), and fusion plasmids were transformed into E. coli NovaBlue (Merck Millipore) according to the manufacturer's instructions. .. The positive recombinants were confirmed by PCR and DNA sequencing and then transformed into E. coli BL21(DE3) (Merck Millipore) for expression.

Article Title: A Sensitive Method for Detecting Zika Virus Antigen in Patients’ Whole-Blood Specimens as an Alternative Diagnostic Approach
Article Snippet: Sequence encoding for amino acid residues 1683–2123 of ZIKV BeH819016 polyprotein (GI:975885966) was codon optimized for Escherichia coli expression, obtained as synthetic DNA (Genscript) and cloned into pET28(a) expression vector. .. Expression was performed in E. coli BL21(DE3) cells (Merck Millipore), using an autoinduction protocol.

Article Title: Discovery of Sanggenon G as a natural cell-permeable small-molecular weight inhibitor of X-linked inhibitor of apoptosis protein (XIAP)
Article Snippet: 2.8 DNA encoding BIR3 of human XIAP protein was amplified by PCR and cloned into the pET30a(+) vector (Merck Millipore, Germany) via the NdeI and SalI restriction sites. .. Protein expression was performed in Escherichia coli BL21(DE3)pLysS (Merck Millipore, Germany).

Article Title: Comparative Transcription Profiling and In-Depth Characterization of Plasmid-Based and Plasmid-Free Escherichia coli Expression Systems under Production Conditions
Article Snippet: Experiments were performed using E. coli BL21(DE3) (Merck KGaA, Darmstadt, Germany). .. BL21(DE3) was transformed with pET30aSOD, yielding BL21(DE3)(pET30aSOD). pET30aSOD was derived by subcloning of the Homo sapiens superoxide dismutase 1 gene (GenBank accession no. ) from pET11aSOD, described elsewhere (SOD, superoxide dismutase) ( ).

Article Title:
Article Snippet: The active concentration of full-length TFPI was determined by titrating a known amount of bovine FXa with TFPI ( ). .. The TFPI constructs TFPI-(1–150) and TFPI-(1–150)-Thr, containing a thrombin cleavage site between K1 and K2 were expressed as insoluble inclusion bodies in Escherichia coli BL21(DE3)pLysS (Merck, Darmstadt, Germany) using the expression vector pET19b (Merck). .. Solubilized (8 m urea, 20 m m DTT, 50 m m Tris-HCl, pH 8.0) TFPI-(1–150)-Thr was folded in 50 m m Tris-HCl (pH 10.0), 1.1 m m oxidized glutathione by rapid dilution followed by dialysis against 20 m m Tris-HCl (pH 7.0).

Article Title: A distal phenylalanine clamp in a hydrophobic channel controls the substrate specificity in the quorum-quenching metallo-γ-lactonase (AiiA) from Bacillus thuringiensis
Article Snippet: Bacillus thuringiensis AHL lactonase (AiiA) was expressed and purified as a TEV (Tobacco Etch Virus) protease cleavable fusion to maltose binding protein according to the procedure reported previously unless noted otherwise ( , ). .. Briefly, E. coli BL21(DE3) cells (Merck, Damstadt, Germany) transformed with the pMAL-t-AiiA vector were grown and induced with 0.5 mM IPTG. .. AiiA was purified as described with an additional gel filtration step ( , ).

Functional Assay:

Article Title: Human Holliday junction resolvase GEN1 uses a chromodomain for efficient DNA recognition and cleavage
Article Snippet: All recombinant proteins were expressed in the E. coli BL21(DE3) pRIL strain (MerckMillipore, Darmstadt, Germany). .. All recombinant proteins were expressed in the E. coli BL21(DE3) pRIL strain (MerckMillipore, Darmstadt, Germany).

Affinity Chromatography:

Article Title: Direct induction of microtubule branching by microtubule nucleation factor SSNA1
Article Snippet: The proteins were expressed in E. coli BL21(DE3) (Merck, Darmstadt, Germany) by inducing with 0.4 mM IPTG (Carl Roth, Karlsruhe, Germany) for overnight at 18 °C. .. Cells were sonicated in lysis buffer (50 mM Na-phosphate buffer pH 7.5, 150 mM NaCl, 10% (v/v) glycerol, 5mM β-mercaptoethanol) supplemented with protease inhibitors (1 mM Pepstatin A, 1mM AEBSF and 1mM Leupeptin) and clarified.

Concentration Assay:

Article Title: Reduction of Spermidine Content Resulting from Inactivation of Two Arginine Decarboxylases Increases Biofilm Formation in Synechocystis sp. Strain PCC 6803
Article Snippet: The E. coli BL21(DE3) strain (Merck Millipore, MA) containing the plasmids was grown in Luria-Bertani (LB) medium containing 100 μg/ml ampicillin overnight at 37°C. .. The E. coli BL21(DE3) strain (Merck Millipore, MA) containing the plasmids was grown in Luria-Bertani (LB) medium containing 100 μg/ml ampicillin overnight at 37°C.

Article Title: Common TFIIH recruitment mechanism in global genome and transcription-coupled repair subpathways
Article Snippet: In brief, p62 or UVSSA was expressed as fusion product with glutathione S-transferase (GST) in a pGEX-4T vector (GE Healthcare) in Escherichia coli BL21(DE3) cells (Merck Millipore). .. In brief, p62 or UVSSA was expressed as fusion product with glutathione S-transferase (GST) in a pGEX-4T vector (GE Healthcare) in Escherichia coli BL21(DE3) cells (Merck Millipore).

Article Title:
Article Snippet: The active concentration of full-length TFPI was determined by titrating a known amount of bovine FXa with TFPI ( ). .. The TFPI constructs TFPI-(1–150) and TFPI-(1–150)-Thr, containing a thrombin cleavage site between K1 and K2 were expressed as insoluble inclusion bodies in Escherichia coli BL21(DE3)pLysS (Merck, Darmstadt, Germany) using the expression vector pET19b (Merck).

Staining:

Article Title:
Article Snippet: Homogeneous human full-length protein was characterized by mass spectrometry for molecular weight determination and SDS-PAGE at reducing and non-reducing conditions followed by Coomassie staining, silver staining, or immunoblotting. .. The TFPI constructs TFPI-(1–150) and TFPI-(1–150)-Thr, containing a thrombin cleavage site between K1 and K2 were expressed as insoluble inclusion bodies in Escherichia coli BL21(DE3)pLysS (Merck, Darmstadt, Germany) using the expression vector pET19b (Merck).

Binding Assay:

Article Title: A distal phenylalanine clamp in a hydrophobic channel controls the substrate specificity in the quorum-quenching metallo-γ-lactonase (AiiA) from Bacillus thuringiensis
Article Snippet: Bacillus thuringiensis AHL lactonase (AiiA) was expressed and purified as a TEV (Tobacco Etch Virus) protease cleavable fusion to maltose binding protein according to the procedure reported previously unless noted otherwise ( , ). .. Briefly, E. coli BL21(DE3) cells (Merck, Damstadt, Germany) transformed with the pMAL-t-AiiA vector were grown and induced with 0.5 mM IPTG.

Variant Assay:

Article Title: Human Holliday junction resolvase GEN1 uses a chromodomain for efficient DNA recognition and cleavage
Article Snippet: Wild type human GEN1 and truncations thereof (residues 2-551, 2-505, 2-464, 2-389) were amplified by PCR from IMAGE clone 40125755 (Mammalian Gene collection, natural variant S92T, S310N, UniProtID Q17RS7) and cloned into a self-made ligation-independent cloning vector with various C-terminal tags followed by His8. .. All recombinant proteins were expressed in the E. coli BL21(DE3) pRIL strain (MerckMillipore, Darmstadt, Germany).

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    Merck KGaA e coli bl21
    SDS-PAGE (a) and western blot (b) analyses of the recombinant BirA protein expressed via pET24-BirA vector in <t>BL21</t> E. coli cells by addition of IPTG to the final concentration of 1 mM. (a) Lane 1: Protein molecular weight marker (Unstained protein molecular weight marker, Fermentas, Lithuania). Different induced colonies (lane 3–7) showed the same size of BirA (36.6 kDa) expression compared with un-induced sample (lane 2) (b) Western blot analysis of recombinant BirA protein by anti-His antibody specific for C-terminal polyhistidine tag indicated the specific 36.6 kDa protein band. Lane 1: Protein marker (PrestainedProtein Ladder, CinnaGen, Iran) Lane 2: Un-induced bacterial lysate. Lane 3: Recombinant BirA protein (36.6 kDa). Arrows indicate the target protein bands
    E Coli Bl21, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 75/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli bl21/product/Merck KGaA
    Average 75 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    e coli bl21 - by Bioz Stars, 2019-12
    75/100 stars
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    92
    Merck KGaA escherichia coli strain bl21
    SDS-PAGE (a) and western blot (b) analyses of the recombinant BirA protein expressed via pET24-BirA vector in <t>BL21</t> E. coli cells by addition of IPTG to the final concentration of 1 mM. (a) Lane 1: Protein molecular weight marker (Unstained protein molecular weight marker, Fermentas, Lithuania). Different induced colonies (lane 3–7) showed the same size of BirA (36.6 kDa) expression compared with un-induced sample (lane 2) (b) Western blot analysis of recombinant BirA protein by anti-His antibody specific for C-terminal polyhistidine tag indicated the specific 36.6 kDa protein band. Lane 1: Protein marker (PrestainedProtein Ladder, CinnaGen, Iran) Lane 2: Un-induced bacterial lysate. Lane 3: Recombinant BirA protein (36.6 kDa). Arrows indicate the target protein bands
    Escherichia Coli Strain Bl21, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/escherichia coli strain bl21/product/Merck KGaA
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    escherichia coli strain bl21 - by Bioz Stars, 2019-12
    92/100 stars
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    SDS-PAGE (a) and western blot (b) analyses of the recombinant BirA protein expressed via pET24-BirA vector in BL21 E. coli cells by addition of IPTG to the final concentration of 1 mM. (a) Lane 1: Protein molecular weight marker (Unstained protein molecular weight marker, Fermentas, Lithuania). Different induced colonies (lane 3–7) showed the same size of BirA (36.6 kDa) expression compared with un-induced sample (lane 2) (b) Western blot analysis of recombinant BirA protein by anti-His antibody specific for C-terminal polyhistidine tag indicated the specific 36.6 kDa protein band. Lane 1: Protein marker (PrestainedProtein Ladder, CinnaGen, Iran) Lane 2: Un-induced bacterial lysate. Lane 3: Recombinant BirA protein (36.6 kDa). Arrows indicate the target protein bands

    Journal: Advanced Biomedical Research

    Article Title: Isolation, cloning, and expression of E. coli BirA gene for biotinylation applications

    doi: 10.4103/2277-9175.161576

    Figure Lengend Snippet: SDS-PAGE (a) and western blot (b) analyses of the recombinant BirA protein expressed via pET24-BirA vector in BL21 E. coli cells by addition of IPTG to the final concentration of 1 mM. (a) Lane 1: Protein molecular weight marker (Unstained protein molecular weight marker, Fermentas, Lithuania). Different induced colonies (lane 3–7) showed the same size of BirA (36.6 kDa) expression compared with un-induced sample (lane 2) (b) Western blot analysis of recombinant BirA protein by anti-His antibody specific for C-terminal polyhistidine tag indicated the specific 36.6 kDa protein band. Lane 1: Protein marker (PrestainedProtein Ladder, CinnaGen, Iran) Lane 2: Un-induced bacterial lysate. Lane 3: Recombinant BirA protein (36.6 kDa). Arrows indicate the target protein bands

    Article Snippet: E. coli BL21 (DE3) and pET-24a(+) plasmid (Merck KGaA, Darmstadt, Germany) were used as expression host and vector, respectively.

    Techniques: SDS Page, Western Blot, Recombinant, Plasmid Preparation, Concentration Assay, Molecular Weight, Marker, Expressing