Bl21 star de3 cells

Manufactured by Thermo Fisher Scientific

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BL21 Star (DE3) cells are a strain of Escherichia coli (E. coli) bacteria commonly used in recombinant protein expression. These cells are designed to enhance the production of recombinant proteins by improving translation efficiency and reducing proteolytic degradation.

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Lab products found in correlation

Hitrap q hp, by GE Healthcare (3 mentions) Hitrap, by Cytiva (3 mentions) Bl21 de3 cells, by Thermo Fisher Scientific (2 mentions) Glutathione sepharose 4b column, by GE Healthcare (2 mentions) Superdex 200 pg, by GE Healthcare (2 mentions) Pgex 6p 3 vector, by GE Healthcare (2 mentions) Pet28a vector, by Merck Group (2 mentions) Superdex 75 prep grade columns, by GE Healthcare (2 mentions) Hiload superdex 200 pg column, by GE Healthcare (2 mentions) Pgex 4t1 vector, by GenScript (2 mentions) Recombinant human tgm2, by R&D Systems (2 mentions) Kmt2a mll1 complex, by Active Motif (2 mentions) Fibrinogen from human plasma, by Merck Group (2 mentions) Ultramer, by Integrated DNA Technologies (2 mentions) Ti e microscope, by Nikon (2 mentions) Ixon3 897 emccd, by Oxford Instruments (2 mentions) Poly d lysine coated glass bottom plates, by MatTek (2 mentions) Ni2 nta affinity column, by Qiagen (1 mentions) Hiload 16 60 superdex 200, by GE Healthcare (1 mentions) Freestyle 293 f cells, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

BL21(DE3) (201 citations) BL21 Star (DE3) (118 citations) BL21(DE3) cells (59 citations) E. coli BL21 Star (DE3) cells (45 citations) BL21 Star (20 citations) Escherichia coli BL21 Star (DE3) cells (14 citations) BL21(DE3)Star-pLysS cells (10 citations) One Shot® BL21 Star™ (DE3) cells (5 citations) BL21 Star (DE3) competent cells (5 citations) E. coli One Shot™ BL21 Star™ (DE3) cells (4 citations)

39 protocols using bl21 star de3 cells

Recombinant Expression and Purification of Nuclear Receptors

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cDNA fragments encoding human TLX LBD (a.a. 187–385) were cloned into pET-46 Ek/LIC vector (Novagen) containing the N-terminal His₆-tag followed by a TEV protease cleavage site according to manufacturer’s protocol and checked by sequencing (ACGT, Inc.). Recombinant protein was expressed in BL21 (DE3) star cells (Life Technologies) using standard methods (induction with 0.1 mM IPTG followed by 24 hours culture at +16°C), and purified using Ni²⁺-NTA affinity column (Qiagen) followed by size exclusion chromatography (HiLoad 16/60 Superdex 200, GE Healthcare, Life Sciences) in buffer containing 20 mM Tris pH 8.0, 150 mM NaCl, 5 mM DTT, 10% glycerol (v/v) and 2 mM CHAPS.
Human estrogen receptor (ER) β His₆-ERβ LBD (residues 261–530) was expressed in BL21 (DE3) cells (Life Technologies) and human LXRβ LBD (Liver X Receptor beta, a.a. 226–460) was expressed from a pET-46 Ek/LIC vector (Novagen) containing the N-terminal His₆-tag followed by TEV protease cleavage site in BL21 (DE3) star cells (Life Technologies). Induction strategy and purification methods were similar to TLX LBD.

Benod C., Villagomez R., Filgueira C.S., Hwang P.K., Leonard P.G., Poncet-Montange G., Rajagopalan S., Fletterick R.J., Gustafsson J.Å, & Webb P. (2014). The Human Orphan Nuclear Receptor Tailless (TLX, NR2E1) Is Druggable. PLoS ONE, 9(6), e99440.

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Expression and Purification of hACE2, hTMPRSS2, and NRP1

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Human ACE2 (1-615aa) with C-terminal polyhistidine and human TMPRSS2 (1-492aa) with C-terminal polyhistidine were expressed in FreeStyle 293F cells (ThermoFisher) with polyethyleneimine (PEI; Sigma-Aldrich). Six days post transfection, the supernatant containing the secreted protein was harvested and syringe-filtered using a 0.45um filter. The filtered supernatant was then incubated with Ni-Sepharose beads (GE healthcare) for 2 h for binding. The beads bound with hACE2 or hTMPRSS2 were then washed and the protein was eluted with 20 mM Tris, 250 mM imidazole, 300 mM NaCl, and buffer were exchanged into 20 mM Tris- HCl, 100 mM NaCl, 2 mM b-mercaptoethanol and 15% glycerol, followed by concentration with Vivaspin 6 spin filters (Sartorius). The purified hACE2 and hTMPRSS2 were stored at −80°C until use.
NRP1-b1(273-427aa) with N terminal polyhistidine was expressed in BL21Star™-(DE3) cells (Invitrogen). Cells were grown at 37°C, and once the cells reached an O.D. value of 0.6, induction was done with 750uM isopropyl β-d-1-thiogalactopyranoside (IPTG) (Sigma). After induction, cells were grown at 22°C for 16 h, following which, they were harvested, centrifuged, lysed and purified using Ni-Sepharose fast flow beads as per stander means described above.

Singh P., Mukherji S., Basak S., Hoffmann M, & Das D.K. (2022). Dynamic Ca2+ sensitivity stimulates the evolved SARS-CoV-2 spike strain-mediated membrane fusion for enhanced entry. Cell Reports, 39(3), 110694.

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Recombinant Expression of Fission Yeast Histones

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The S. pombe histones spH2A, spH2B, and spH3 cDNAs were cloned into pET-13 plasmids, and the transformation was done in E.coli strains BL21 (DE3) pLyS (Stratagene). The transformation for spH4 plasmid was done in E.coli strains BL21 star (DE3) cells (Invitrogen). Transformed colonies were selected based on Kanamycin resistance. Individual bacterial colonies containing the respective histone gene plasmids were, inoculated in 3 mL LB media having 34 mg/mL Kanamycin and 34 mg/mL chloramphenicol and incubate at 37 °C until OD = 0.3 to 0.4. For histone H4, colonies were inoculated in 10 mL LB media + 12.5 mM MgCl₂ and MgSO₄, incubated at 37 °C until OD = 0.6. Bacterial cells were induced with 0.5 mM IPTG and incubated for 2.5 to 3 h at 37 °C. 1 mL of each culture was harvested, cells were re-suspended in loading buffer and sonicated. The samples were heated at 95 °C for 2 min and run in 18% SDS-PAGE gels to check histone expression and to select the best-expressing colonies for large-scale induction. Large cultures were inoculated in 12 L for spH4 and 6 lit for spH2A, spH2B, and spH3) from the pre-culture of the chosen colonies. Media compositions and IPTG induction were the same as for the mini-expression cultures. The cells are harvested and stored at −80 °C for protein extraction.

Dong W., Prasad P., Lennartsson A, & Ekwall K. (2021). The Role of Non-Catalytic Domains of Hrp3 in Nucleosome Remodeling. International Journal of Molecular Sciences, 22(4), 1793.

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Recombinant Mouse Prion Protein Production

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Starting with the full length wild type mouse prion protein in the pJ414 vector backbone (DNA 2.0), we used QuickChange site-directed mutagenesis PCR to construct mutants, with primers generated manually and by www.primerdesigner.com. DNA was transformed into E. coli BL21Star (DE3) cells (Invitrogen) and expressed in M9 minimal media supplemented with 1 g/L ¹⁵N ammonium chloride (Cambridge Isotope Labs). Cells were grown at 37 °C until they reached an OD600 of 1.0, at which point the temperature was dropped to 30 C and protein expression induced with 1 mM isopropyl-1-thio-D-galactopyranoside (IPTG). Cells were allowed to express for 20 hours. Inclusion bodies were washed, then treated with 8 M guanidine HCl in pH 8 tris-acetate buffer to extract proteins. The extracted proteins were purified by immobilized metal affinity chromatography, allowed to fold overnight at pH 8, then transferred into pH 4.5 sodium acetate buffer by size exclusion chromatography. The proteins were then dialyzed into Milli-Q ultrapure water and purified further by reverse phase HPLC using C4 silica resin. Identity and purity were checked using a Sciex X500b mass spectrometer. The proteins were lyophilized, and quantified by UV-Vis spectrophotometry before use.

Schilling K.M., Tao L., Wu B., Kiblen J.T., Ubilla-Rodriguez N.C., Pushie M.J., Britt R.D., Roseman G.P., Harris D.A, & Millhauser G.L. (2020). Both N-Terminal and C-Terminal Histidine Residues of the Prion Protein are Essential for Copper Coordination and Neuroprotective Self-Regulation.. Journal of molecular biology, 432(16), 4408-4425.

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Purification of Pf LipL1 Protein Variants

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All constructs were transformed into BL21-Star (DE3) cells (Invitrogen) containing the pRIL plasmid isolated from BL21-CodonPlus-RIL cells (Agilent) and plasmid pRK586 encoding the Tobacco Etch Virus (TEV) protease as described.^{17 (link)} These cells produce a protein product fused to an amino-terminal hexahistidine tag. 2L of TB media containing ampicillin, kanamycin, and chloramphenicol were inoculated with an overnight culture for an initial OD₆₀₀ of approximately 0.1. The cells were grown to mid log phase at 37°C and then the temperature was reduced to 20°C. Protein expression was induced with 0.4 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) and the cells were harvested after 10h. Purification was performed as described previously for full-length PfLipL1.¹⁴ Briefly, PfLipL1 variants were purified by immobilized metal ion chromatography followed by cation exchange chromatography and gel filtration chromatography. The first two steps of purification were performed on the same day to avoid proteolytic cleavage. Purified PfLipL1 mutants were concentrated to approximately 5 mg·mL⁻¹ and stored at −80°C.

Guerra A.J., Afanador G.A, & Prigge S.T. (2017). Crystal structure of lipoate-bound lipoate ligase 1, LipL1, from Plasmodium falciparum. Proteins, 85(9), 1777-1783.

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Purification of Recombinant NBC Protein

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Full-length coding sequence of NBC was introduced into pEASY-Blunt E2-His-MBP vector. The pEASY-Blunt E2-His-MBP-NBC plasmid was transformed into E. coli BL21 Star (DE3) cells (Invitrogen). Cell culture was grown in LB (lysogeny broth) medium to an OD_600nm of 0.6 at 37 °C and subsequently transferred to 18 °C. After 30 min of incubation, protein expression was induced by isopropyl-β-D-thiogalactoside (0.5 mM). After 12 h of incubation, the cultures were harvested by centrifugation (4000 rpm) at 4 °C. E. coli pellets containing recombinant proteins were lysed by sonication in lysis buffer (1 mM DTT, 50 mM Tris-HCl (pH 7.4), 300 mM NaCl, 1 tablet EDTA-free protease Inhibitor cocktail per 50 ml (Roche). The cell extracts were applied on a Ni-TED 1 ml Sefinose (TM) Column (Sangon Biotech, https://www.sangon.com/). The purity of the fractions corresponding to purified monomeric recombinant proteins was analyzed on Coomassie-stained SDS-polyacrylamide gel electrophoresis (SDS–PAGE) gels and the protein concentrations were determined by OD_280nm measurements.

Liu H.Q., Pu Z.X., Di D.W., Zou Y.J., Guo Y.M., Wang J.L., Zhang L., Tian P., Fei Q.H., Li X.F., Khaskheli A.J., Wu L, & Guo G.Q. (2022). Significance of NatB-mediated N-terminal acetylation of auxin biosynthetic enzymes in maintaining auxin homeostasis in Arabidopsis thaliana. Communications Biology, 5, 1410.

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Purification of α-Catenin and Vinculin

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α-Catenin and vinculin were expressed and purified as reported in our previous study^{26 (link)}. DNA fragments of mouse αE-catenin M₁-M₃ (residues 276–634) and M₂-M₃ (residues 385–634) were amplified by PCR and cloned into the pGEX6P-3 vector (GE Healthcare). A DNA fragment of mouse full-length vinculin (residues 1–1066) was cloned into the pET-6b (+) vector (Novagen). These plasmids were verified by DNA sequencing and transformed into Escherichia coli strain BL21Star (DE3) cells (Invitrogen). α-Catenin and vinculin molecules were expressed at 20 °C in Luria-Bertani medium supplemented with 0.1 mM isopropyl-β-d-thiogalactopyranoside. BL21Star cells expressing α-catenin and vinculin were suspended in 20 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl and disrupted by sonication. The supernatant after ultracentrifugation was applied to a Glutathione Sepharose 4B column (GE Healthcare). Proteins eluted from the column were further purified by anion exchange (HiTrap Q HP, GE Healthcare) and gel filtration (Superdex 200 pg, GE Healthcare) chromatography. N-terminal His₆ tags on vinculin molecules were then cleaved using human rhinovirus 3C protease. For AFM structural imaging, GST-tags at N-termini of the wild-type and mutated fragments were cleaved by PreScission Protease (GE Healthcare).

Maki K., Han S.W., Hirano Y., Yonemura S., Hakoshima T, & Adachi T. (2018). Real-time TIRF observation of vinculin recruitment to stretched α-catenin by AFM. Scientific Reports, 8, 1575.

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Recombinant MIF Production in E. coli

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rMIF protein was produced in E. coli BL21 Star (DE3) cells (Invitrogen), after induction with 1mM IPTG for 3.5 hours as in [17 (link)]. Bacterial cells were pelleted by centrifugation in a Beckman Allegra 6R centrifuge (3,000 rpm, 15 min, ambient temperature). Approximately 1g of bacterial pellet was resuspended in 5 ml of 50 mM Tris pH 8.5, 50 mM KCl, 5mM magnesium acetate, 0.1% sodium azide, and sonicated. The lysate was centrifuged at 15,000 rpm for 20 minutes, then filtered through Millipore Steriflip vacuum filtration system with 0.2 µM membranes. The filtered lysate was loaded onto a 5 mL QHP HiTrap anion exchange column in a negative chromatography mode such that rMIF remained unbound. The rMIF was buffer exchanged and concentrated by loading onto Mono S cation exchange column and eluted with a linear gradient.

Dwyer M., Shan Q., D'Ortona S., Maurer R., Mitchell R., Olesen H., Thiel S., Huebner J, & Gadjeva M. (2014). Cystic Fibrosis Sputum DNA Has NETosis Characteristics and Neutrophil Extracellular Trap Release Is Regulated by Macrophage Migration-Inhibitory Factor. Journal of Innate Immunity, 6(6), 765-779.

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Profiling Neem Triterpenoids by UPLC-MS

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Neem tissues for the profiling of triterpenoids were collected from Pune region, Maharashtra, India in the period March to May. Fifteen reference triterpenoids were isolated and characterized as reported earlier [53 (link), 54 (link), 6 (link)] and described briefly in Additional file 1. For extraction, HPLC grade solvents were purchased from Sigma (St. Louis, MO, USA). For UPLC-ESI(+)-MS experiments LC-MS grade solvents were procured from Avantor Performance Materials, JT Baker (PA, USA). SuperScript® III First-Strand Synthesis System (Invitrogen) was used for cDNA synthesis. For PCR amplification, AccuPrime™ (Invitrogen) polymerase was used. For Restriction digestion, NEW ENGLAND BioLabs®_inc(NEB) restriction enzymes were used. Gel extraction of restricted product and vector were carried out by GenElute™ Gel Extraction Kit from Sigma. T₄ DNA ligase from Invitrogen was used for ligation. TOP10 cells (Invitrogen) were used for cloning. Lemo21 (DE3) cells (NEB), BL21 (DE3) cells (NEB) and BL21 Star (DE3) cells (Invitrogen) were used as expression cells. Ni-NTA agarose (Invitrogen) was used for protein purification. Enzyme assay samples were analyzed on Agilent 7890A GC coupled with 5975C mass detector. Geraniol, nerol, (E,E)-farnesol, squalene standards were purchased from Sigma Aldrich. IPP, FPP, GPP, and DMAPP were synthesized as reported previously [55 (link), 56 ].

Pandreka A., Dandekar D.S., Haldar S., Uttara V., Vijayshree S.G., Mulani F.A., Aarthy T, & Thulasiram H.V. (2015). Triterpenoid profiling and functional characterization of the initial genes involved in isoprenoid biosynthesis in neem (Azadirachta indica). BMC Plant Biology, 15, 214.

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Isotopic Labeling and Purification of CheY

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A plasmid containing E. coli CheY was provided by Dr. Robert Bourret (University of North Carolina at Chapel Hill) and was subcloned into the pET28a plasmid (Novagen). All mutants were prepared by site-directed mutagenesis. The CheY vector was transformed into BL21 Star (DE3) cells (Invitrogen) and grown on M9 minimal media with the appropriate isotope(s): ¹⁵NH₄Cl (99%) and/or D-glucose (U-¹³C₆−99%) as the sole nitrogen and carbon sources, respectively. Cells were grown at 37 °C and induced with 1 mM isopropyl 1-thio-β-d-galactopyranoside when the OD₆₀₀ reached 0.6 and were grown for an additional 22–26 h at 20 °C. The cells were harvested by centrifugation, resuspended in buffer A (25 mM Tris, 10 mM MgCl₂, pH 8.0), and sonicated. The lysate was then centrifuged at 6000 r.p.m. and dialyzed overnight into buffer A at 4 °C. The protein was purified on a Q-Sepharose Fast Flow column (GE Healthcare) equilibrated with buffer A and eluted in buffer B (buffer A with the addition of 1.5 M NaCl. Q-Sepharose purified protein was passed over a G75 superdex gel-filtration column equilibrated with NMR buffer (50 mM NaP_i, 0.02% NaN₃, pH 7.0 and an appropriate amount of MgCl₂ and/or EDTA). The CheY elution peak was concentrated for further experiments.

Wang J., Jain A., McDonald L.R., Gambogi C., Lee A.L, & Dokholyan N.V. (2020). Mapping allosteric communications within individual proteins. Nature Communications, 11, 3862.

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