E coli nico21 de3

Manufactured by New England Biolabs

Sourced in United States

E. coli NiCo21(DE3) is a competent E. coli cell line designed for recombinant protein expression. It is engineered to facilitate the expression and purification of proteins containing a polyhistidine tag.

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Lab products found in correlation

Xtractor buffer, by Takara Bio (1 mentions) Capturem his tagged purification kit, by Takara Bio (1 mentions)

Close spelling variants of this product

E. coli NiCo21 (DE3) cells E. coli strain NiCo21(DE3) NiCo21(DE3)

4 protocols using e coli nico21 de3

Cloning and Mutagenesis of N. meningitidis Proteins

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The genes were PCR amplified from genomic DNA isolated from N. meningitidis MC58 using the appropriate primers (S2 Table). The PCR product was digested with the restriction enzymes XbaI and SalI and ligated into a identically digested pET-28b(+) vector (Novagen). The resulting plasmid pSB13 coding for a protein with a C-terminal 6xHis-tag was transformed into E. coli NiCo21 (DE3) (NEB) containing the plasmid pLysS (Novagen). The point mutation in the ATP binding motif (K72A) was created in pSB13 using site-directed mutagenesis with the primers SF177 and SF178 resulting into pSAF92. Cloning of SSB_Nm has been described earlier [74 (link)]. Primers SF275 and SF276 were used to amplify the vector pSAF104 using the vector pEH1 as a template to obtain the ssb_NmΔ8C construct.
The sequences of all constructs were verified using appropriate sequencing primers.

Frye S.A., Beyene G.T., Namouchi A., Gómez-Muñoz M., Homberset H., Kalayou S., Riaz T., Tønjum T, & Balasingham S.V. (2017). The helicase DinG responds to stress due to DNA double strand breaks. PLoS ONE, 12(11), e0187900.

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Recombinant Expression and Purification of Dianthin 30 and Gypsophilin-S

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N-terminally His-tagged dianthin 30 (His-dianthin) from the plant Dianthus caryophyllus L. was recombinantly expressed in E. coli NiCo21(DE3) (New England Biolabs, Ipswich, QLD, USA), purified by Ni-nitrilotriacetic acid affinity chromatography and analyzed by SDS-PAGE as described elsewhere [42 (link)]. Gypsophilin-S was isolated from the seeds of Gypsophila elegans M.Bieb. using ammonium sulfate precipitation and subsequent ion exchange chromatography. The isolation is described in detail elsewhere [29 (link)].

Schlaak L., Weise C., Kuropka B, & Weng A. (2022). Sapovaccarin-S1 and -S2, Two Type I RIP Isoforms from the Seeds of Saponaria vaccaria L.. Toxins, 14(7), 449.

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Functional Analysis of Arginine Kinase Mutants

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For functional analysis of the residues Met⁷⁰ and Met¹⁷⁹ in rmCK, two recombinant single mutants (Met⁷⁰Ala and Met¹⁷⁹Leu) and one recombinant double mutant (Met⁷⁰Ala/Met¹⁷⁹Leu) were produced (Proteros biostructures, Martinsried, Germany). Three plasmids inducing the desired mutations were synthesized by Geneart (Regensburg, Germany): (i) HIS6-TEV-rabbitCK(1–381)-Met179Leu, (ii) HIS6-TEV-rabbitCK(1–381)-Met70Ala, and (iii) HIS6-TEV-rabbitCK(1–381)-Met70Ala/Met179Leu. Plasmids were transformed in E. coli NiCo21(DE3) obtained from New England Biolabs (Ipswich, MA, USA) for rmCK protein expression.

Steinritz D., Lüling R., Siegert M., Mückter H., Popp T., Reinemer P., Gudermann T., Thiermann H, & John H. (2021). Alkylation of rabbit muscle creatine kinase surface methionine residues inhibits enzyme activity in vitro. Archives of Toxicology, 95(10), 3253-3261.

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Protein Purification and Nuclease Assay

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The full-length qnu and its truncated allele, Δqnu, missing the N-terminal 30% encoding the DNA-binding QN domain, were PCR amplified using the following primers: qnuR (5’-GAGGTACCTGGATTAATATAATTTTATGGTCGAGGAG-3’), and qnuF1 (5’- AGGGATCCATGAATTATAATCATACTGGTCAATATAAAAC-3’) or qnuF2 (5’- AGGGATCCATGTTACCTCATAATAATAATCTTCCTAATTT-3’), respectively. Upon cleavage with KpnI and BamHI, the amplified products were cloned at the KpnI-BamHI site of the pProEx THb vector (ThermoFisher, Pittsburgh, PA).
The recombinant plasmids were introduced into E. coli NiCo21(DE3) (New England Biolabs, Ipswich, MA). Bacteria were grown to the exponential phase, at which point the expression of recombinant proteins was induced with 1 mM IPTG at 30 °C for 6 h. Upon harvesting, the cells were disrupted using xTractor Buffer (Clontech). Recombinant proteins were purified using a CapturemHis-Tagged Purification Kit (Clontech). For the nuclease activity assay, 20 ng of protein was incubated with 400 ng pGEM-derived plasmid in a 20 μl reaction mixture containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 2 mM MgCl₂ at 37 °C for 2 h. The samples were then electrophoresed in 1% agarose gel with ethidium bromide and analyzed under UV light.

, & Szafranski P. (2017). Evolutionarily recent, insertional fission of mitochondrial cox2 into complementary genes in bilaterian Metazoa. BMC Genomics, 18, 269.

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