Why express in eukaryotic systems— if you can achieve higher yields and activity in E. coli?
BL21 and Beyond Novagen first commercialized BL21 in 1990 and it has remained the gold standard among expression hosts ever since. Being deficient in both lon and ompT proteases, BL21 and its derivatives are the most popular hosts today. Yet, a number of other Novagen host strains are available, as lDE3 lysogens for T7 promoter-driven expression, and as non-lDE3 lysogens for expression from E. coli promoters. These state-of-the-art strains are powerful tools for situations more challenging than BL21 can support, particularly the expression of mammalian proteins in E. coli. This online spotlight guide can help you select the best host strain for your protein and your application.
Rosetta™ Technology
The first bacterial host system to offer "universal" translation by supplementing six or seven rare tRNAs in one strain
Rosetta 2 Competent Cells
are designed to enhance the expression of eukaryotic proteins that contain codons rarely used in E. coli
supply tRNAs for seven rare codons (AUA, AGG, AGA, CUA, CCC, CGG, and GGA) on a compatible chloramphenicol-resistant plasmid
provide for "universal" translation compared with native E. coli
offer the Rosetta 2 (DE3)pLysS and Rosetta 2 (DE3)pLacI derivatives, which contain the rare tRNA genes on the same plasmids that carry the T7 lysozyme and lac repressor genes, respectively
are derived from BL21, deficient in both lon and ompT proteases for enhanced protein stability
Map of pRARE2 plasmid family The basic structure of pRARE2 is indicated. pLysSRARE and pLacIRARE (positioned between the p15a ori and the tRNA gene for ileX) contain the genes encoding T7 lysozyme (lysS) and lac repressor (lacI), respectively. Also indicated are chloramphenicol resistance gene (Cam), replicon (p15a ori) and tRNA genes. tRNA genes corresponding to rare codons in E. coli are indicated in blue.
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RosettaBlue™ Competent Cells
have all the rare tRNA genes of the basic Rosetta strain
are a NovaBlue derivative that also features high transformation efficiency and recA, endA, and lacIq mutations
have trxB/gor mutations for disulfide bond formation and improved protein folding in vivo
Rosetta-gami B Competent Cells
combine the advantages of Origami B and Rosetta strains in one host
have trxB/gor mutations for disulfide bond formation and improved protein folding in vivo
are derived from a lacZY mutant of BL21 to enable precise control of expression levels by adjusting the concentration of IPTG
include the lon and ompT deficiencies of BL21 which increase protein stability
Expression of D6-175 vtPA in different host strains vtPA constructs in pET-21d(+) and pTriEx-3 were transformed into the indicated host strains. Cultures were grown at 37°C in LB + 0.5% glucose to an OD600 of 0.6 to 1.0 and aliquots induced with 1 mM IPTG for 3 h. Total cell protein samples were prepared and then analyzed by SDS-PAGE (4–20% gradient gels) and Coomassie blue staining. Panel A, pET-21d(+) vtPA; Panel B, pTriEx-3 vtPA. Duplicate induced cultures are shown in Panel B.
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Origami™ Technology
Exclusive host strains that promote disulfide bond formation and increase protein solubility and activity (1, 2, 3, 4)
provide mutations in both the thioredoxin reductase (trxB) and glutathione reductase (gor) genes, greatly enhancing disulfide bond formation in the cytoplasm
permit protein folding in bacterial cytoplasm (1, 2)
are ideal for use with pET-32 vectors, since the thioredoxin fusion tag further enhances the formation of disulfide bonds in the cytoplasm
Origami 2 Competent Cells
new generation strains are kanamycin-sensitive strains compatible with kanamycin-resistant vectors
K-12 derivatives
Bacterial host influences on solubility of target protein and enzyme activity4
A. Solubility of recombinant chitinase expressed in pET-22 vectors using BL21(DE3) and Origami(DE3) E. coli strains Hosts were grown to OD600 of 0.6 to 1.0. After induction with 1 mM IPTG, culture growth was continued for 4 h at 37°C or for 24 h at 20°C. Samples of the total (T), soluble (S), and insoluble (I) protein fractions were prepared and analyzed by 15% SDS-PAGE and Coomassie blue staining.
B. CD spectra CD spectra of a recombinant rye seed chitinase expressed in Origami E. coli compared to the native form5.
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C. Chitinase activity of the original and recombinant chitinases toward glycolchitin
Enzyme
Specific activity (units/mg)
RSC-c
205.0
rRSC-c
233.7
Origami 2 Competent Cells
new generation strains are kanamycin-sensitive strains compatible with kanamycin-resistant vectors
K-12 derivatives
Origami B Competent Cells
derived from a lacZY mutant of BL21 to enable precise control of expression levels by adjusting the concentration of IPTG
include the lon and ompT deficiencies of BL21 which increase protein stability
Rosetta-gami, Rosetta-gami 2, and Rosetta-gami B Competent Cells
Lobel, L., Pollak, S., Klein, J., and Lustbader, J. W. (2001) Endocrine14(2), 205–212.
Lobel, L., Pollak, S., Lustbader, B., Klein, J. and Lustbader, J. W. (2002) Protein Express. Purif.25(1), 124–133.
Prinz, W. A., Aslund, F., Holmgren, A., and Beckwith, J. (1997) J. Biol. Chem.272, 15661–15667.
Data provided by Takayuki Ohnuma1, Mikako Yagi2, Toki Taira2, Takeshi Yamagami2, and Masatsune Ishiguro2 1University of Illinois at Urbana-Champaign, USA 2Kyushu University, Japan
Imoto, T. and Yagishita, K. (1971) Agric. Biol. Chem.35, 1154–1156.
