E coli rosetta 2 de3 cells

Manufactured by Merck Group

E. coli Rosetta 2(DE3) cells are a strain of Escherichia coli bacteria genetically modified for protein expression. They are designed to enhance the expression of proteins that contain codons rarely used in E. coli.

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Lab products found in correlation

Qiaexpressionist handbook, by Qiagen (1 mentions) Pet 22b plasmid, by Merck Group (1 mentions) Phusion dna polymerase, by Thermo Fisher Scientific (1 mentions) T4 dna ligase, by New England Biolabs (1 mentions)

Close spelling variants of this product

Rosetta 2(DE3) competent E. coli cells E. coli Rosetta 2(DE3) Singles Competent Cells E. coli Rosetta 2(DE3) pLysS cells E. coli BL21 (DE3) Rosetta 2 cells E. coli Rosetta 2 (DE3) E. coli Rosetta 2 cells E. coli Rosetta cells Rosetta 2(DE3) cells Rosetta (DE3) cells Rosetta 2(DE3)

3 protocols using e coli rosetta 2 de3 cells

Recombinant Cox19 protein expression

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The coding sequence without the stop codon of Cox19 and Cox19^EE was amplified from pRS426-Cox19 or Cox19^EE and cloned into pET22b (Novagen) upstream of a hexahistidine sequence using the restriction sites BamHI and XhoI. These plasmids were transformed into E. coli Rosetta 2(DE3) cells (Merck, Billerica, MA). The cells were grown at 37°C to an OD₆₀₀ of 0.6 before expression was induced by the addition of 1 mM isopropyl-β-d-thiogalactoside (IPTG) for 4 h at 37°C. The native purification of the recombinant proteins was performed as described in protocols 9 and 12 of the QIAexpressionist Handbook (Qiagen, Venlo, Netherlands).

Bode M., Woellhaf M.W., Bohnert M., van der Laan M., Sommer F., Jung M., Zimmermann R., Schroda M, & Herrmann J.M. (2015). Redox-regulated dynamic interplay between Cox19 and the copper-binding protein Cox11 in the intermembrane space of mitochondria facilitates biogenesis of cytochrome c oxidase. Molecular Biology of the Cell, 26(13), 2385-2401.

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Heterologous Expression of AngCDA in Pichia

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The AngCDA gene (UniProt ID: A2QZC8), without the sequence encoding the 19 amino acid signal peptide, was codon optimized for Pichia pastoris and synthesized by GeneArt. It was amplified with corresponding overlaps to be cloned via Gibson assembly (71 (link)) into a previously generated pET-22b(+) plasmid (Merck KGaA), already containing either an N-terminal pelB and a C-terminal Strep-tag II sequence or both an N- and C- terminal Strep- tag II. Both constructs were transformed into E. coli Rosetta™ 2 (DE3) cells (Merck KGaA) for protein expression.

Bonin M., Hameleers L., Hembach L., Roret T., Cord-Landwehr S., Michel G, & Moerschbacher B.M. (2021). In silico and in vitro analysis of an Aspergillus niger chitin deacetylase to decipher its subsite sugar preferences. The Journal of Biological Chemistry, 297(4), 101129.

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Cloning and Expression Optimization

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Target genes were amplified using Phusion® DNA polymerase (Thermo). Genes were ligated into the pETcc2 (Peat et al., 2013 (link)) (modified pET14b, Novagen) expression vector using NdeI and BamHI restriction endonucleases and T4 DNA ligase (New England Biolabs). All proteins were expressed and purified from either E. coli BL21 (DE3), purchased from Novagen, or in E. coli Rosetta 2 (DE3) cells, purchased from EMD Millipore. All chemicals were ordered from Sigma Aldrich (St Louis, MO, USA). Synthetic genes were purchased from GeneART (Invitrogen).

Wilding M., Walsh E.F., Dorrian S.J, & Scott C. (2015). Identification of novel transaminases from a 12-aminododecanoic acid-metabolizing Pseudomonas strain. Microbial Biotechnology, 8(4), 665-672.

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