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E. coli

NEB offers two protein expression systems in E.coli. The pMAL™ Protein Fusion & Purification System (NEB #E8200) is used to express an MBP-fusion protein which is then purified by affinity purification. This system enhances solubility and results in reliable E.coli expression in either the cytoplasm or periplasm.

The IMPACT™ Kit (NEB #E6901) allows fusion of a tag consisting of the intein and the chitin binding domain (CBD), to either the C-terminus (pTXB1) or the N-terminus (pTYB21) of the target protein. In the presence of thiols, such as DTT, the intein undergoes specific self-cleavage which releases the target protein from the chitin-bound intein tag resulting in purification in a single chromatographic step with no need for proteases.

pMAL™ and IMPACT™ are trademarks of New England Biolabs, Inc.

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E. coli includes these subcategories:
SHuffle Expression Strains
FAQs for E. coli
Protocols for E. coli
Application Notes for E. coli
    Publications related to E. coli
  1. Guo, C., et al. 2004. Intein-mediated fusion expression, high efficient refolding, and one-step purification of gelonin toxin Protein Expr Purif.. 37, PubMedID: , DOI:
  2. Grzybowska, B., Szweda, P. and Synowiecki, J. 2004. Cloning of the thermostable alpha-amylase gene from Pyrococcus woesei in Escherichia coli: isolation and some properties of the enzyme Mol. Biotechnol. . 26, PubMedID: 14764935, DOI:
  3. Lue, R.Y., et al. 2004. Versatile protein biotinylation strategies for potential high-throughput proteomics J Am. Chem. Soc.. 126, PubMedID: 14746473, DOI:
  4. Pezza, J.A., Allen, K.N. and Tolan, D.R. 2004. Intein-mediated purification of a recombinantly expressed peptide Chem Commun (Camb). 21, PubMedID: 15514791, DOI:
  5. Takeda, S.,et al. 2004. Site-specific conjugation of oligonucleotides to the C-terminus of recombinant protein by expressed protein ligation Bioorg. Med. Chem. Lett.. 14(10), PubMedID: 15109622, DOI:
  6. Romanelli, A., et al. 2004. Semisynthesis of a segmental isotopically labeled protein splicing precursor: NMR evidence for an unusual peptide bond at the N-extein-intein junction Proc. Natl. Acad. Sci. USA. 101, PubMedID: 15087498, DOI:
  7. Hemelaar, al. 2004. Specific and covalent targeting of conjugating and deconjugating enzymes of ubiquitin-like proteins Mol. Cell Biol.. 24, PubMedID: 14673145, DOI:
  8. Durek, T., et al. 2004. Synthesis of fluorescently labeled mono- and diprenylated Rab7 GTPase J. Am. Chem. Soc.. 126, PubMedID: 15600338, DOI:
  9. Demidov, V.V., and Broude, N.E. 2006. Profluorescent protein fragments for fast bimolecular fluorescence complementation in vitro Nat. Protoc.. 1, PubMedID: 17406301, DOI:
  10. Durek T., Goody R.S and Alexandrov K. 2004. In vitro semisynthesis and applications of C-terminally modified Rab proteins Methods Mol Biol.. 283, PubMedID: 15197315, DOI:
  11. Kalia, J., et al. 2006. Reactivity of intein thioesters: appending a functional group to a protein Chembiochem.. 7, PubMedID: 16897799, DOI:
  12. Eckenroth, B., et al. 2006. Semisynthesis and characterization of mammalian thioredoxin reductase Biochem.. 45, PubMedID: 16618105, DOI:
  13. Grant, J., et al. 2006. The N terminus of GTP gamma S-activated transducin alpha-subunit interacts with the C terminus of the cGMP phosphodiesterase gammasubunit J. Biol. Chem. . 281, PubMedID: 16407279, DOI:
  14. Gottlieb, D., et al. 2006. Intein-mediated in vitro synthesis of lipidated Ras proteins Chem. Commun. (Camb). 21, PubMedID: 16391727, DOI:
  15. Esipov, R.S., et. al. 2006. Production and purification of recombinant human glucagons overexpressed as intein fusion protein in Escherichia coli Protein Pept. Lett.. 13, PubMedID: 16712508, DOI:
  16. Wojtaszek, J., et al. 2006. LT CI, a novel chymotrypsin inhibitor of the potato I family from the earthworm Lumbricus terrestris. Purification, cDNA cloning, and expression Comp. Biochem. Physiol B. Biochem. Mol. Biol. . 143, PubMedID: , DOI:
  17. Lee, N.Y., et al. 2006. Structure and dynamics of the epidermal growth factor receptor C-terminal phosphorylation domain Protein Sci.. 15, PubMedID: 16597832, DOI:
  18. Hackenberger, C. P., et. al. 2006. Expression of N-terminal Cys-protein fragments using an intein refolding strategy Bioorg. Med. Chem.. 14, PubMedID: , DOI:
  19. Ge, X., et. al. 2005. Self-cleavable stimulus responsive tags for protein purification without chromatography J. Am. Chem. Soc. 127, PubMedID: 16089436, DOI:
  20. Girish, A., et al. 2005. Site-specific immobilization of proteins in a microarray using intein-mediated protein splicing Bioorg. Med. Chem. Lett.. 15, PubMedID: 15863295, DOI:
  21. Goodin, J. L., et al. 2005. Yersinia pestis outer membrane type III secretion protein YscC: expression, purification, characterization, and induction of specific antiserum Protein Expr. Purif. . 40, PubMedID: 15721783, DOI:
  22. Rebets, Y., et al. 2005. DNA -binding activity of LndI protein and temporal expression of the gene that upregulates landomycin E production in Streptomyces globisporus 1912 Microbiology . , PubMedID: , DOI:
  23. Morassutti, C., et al. 2005. Expression of SMAP-29 cathelicidin-like peptide in bacterial cells by intein-mediated system Protein Expr. Purif.. 39, PubMedID: , DOI:
  24. Ingham, A.B., et al. 2005. A versatile system for the expression of nonmodified bacteriocins inEscherichia coli J. Appl. Microbiol. . 98, PubMedID: 15715871, DOI:
  25. Lu, M., et. al. 2010. Purification of untagged HIV-1 reverse transcriptase by affinity chromatography Protein Expr. Purif.. 71, PubMedID: 20060474, DOI:
  26. Chen, J., et al. 2010. Chemically ubiquitylated PCNA as a probe for eukaryotic translesion DNA synthesis Nat. Chem. Biol. . 6, PubMedID: 20208521, DOI:
  27. Karukurichi, K. et al. 2010. Analysis of p300/CBP histone acetyltransferase regulation using circular permutation and semisynthesis J. Am. Chem. Soc. . 132, PubMedID: 20063892, DOI:
  28. Collins, E. D., et. al. 2010. Cloning the human vitamin D receptor into the pTwin-1 expression vector J Steroid Biochem Mol Biol. , PubMedID: 20171280 , DOI:
  29. Manconi, B., et. al. 2010. Expression, purification, phosphorylation and characterization of recombinant human statherin Protein Expr Purif.. 69, PubMedID: , DOI:
  30. Yu, H. H., et. al. 2010. Expressed protein ligation for the preparation of fusion proteins with cell penetrating peptides for endotoxin removal and intracellular delivery Biochim Biophys Acta . , PubMedID: 20170629, DOI:
  31. Lu, X., et al. 2010. Designed semisynthetic protein inhibitors of ub/ubl e1 activating enzymes J. Am. Chem. Soc. . 132, PubMedID: 20099854, DOI:
  32. Berkmen, M. 2012. Production of disulfide-bonded proteins in Escherichia coli Protein Expression and Purification. , PubMedID: 22085722, DOI:
  33. Hemmis, C.W., Berkmen, M., Eser, M.and Schildbach, J.F. 2011. TrbB from conjugative plasmid F is a structurally distinct disulfide isomerase that requires DsbD for redox state maintenance. J Bacteriol. 193(18), PubMedID: 21742866, DOI: 10.1128/JB.00351-11
  34. Zhao, W. et al. 2008. An efficient on-column expressed protein ligation strategy: application to segmental triple labeling of human apolipoprotein E3 Protein Sci. . 17, PubMedID: 18305193, DOI:
  35. Zhao, Z., et. al. 2008. Purification of green fluorescent protein using a two-intein system Appl. Microbiol. Biotechnol.. 77, PubMedID: 17973109, DOI:
  36. Skrisovska, L. et al. 2008. Improved segmental isotope labeling methods for the NMR study of multidomain or large proteins: application to the RRMs of Npl3p and hnRNP L. J. Mol. Biol.. 375, PubMedID: 17936301, DOI:
  37. Ottesen, J. et al. 2008. An amalgamation of solid phase peptide synthesis and ribosomal peptide synthesis Biopolymers. 90, PubMedID: 17636509, DOI:
  38. Chen, Y. Q., et. al. 2008. Expression of a cytotoxic cationic antibacterial peptide in Escherichia coli using two fusion partners Protein Exp. Purif.. 57, PubMedID: 17977015, DOI:
  39. Babu, S.K., et. al. 2008. Construction of intein mediated hGMCSF expression vector and its prufication in Pichia pastoris Protein Exp. Purif.. 57, PubMedID: 18309571, DOI:
  40. Anton, B.P., Fomenkov, A., Raleigh, E.A. and Berkmen, M. 2016. Complete Genome Sequence of the Engineered Escherichia coli SHuffle Strains and Their Wild-Type Parents Genome Announc.. Mar 31;4(2), PubMedID: 27034504, DOI: 10.1128/genomeA.00230-16.
  41. Nakano, S., et al. 2002. Multiple pathways of Spx (YjbD) proteolysis in Bacillus subtilis J. Bacteriol. 184, PubMedID: 12057962, DOI:
  42. Zhong, Q., et al. 2002. Endosomal localization and function of sorting Proc. Natl. Acad. Sci. USA. 99, PubMedID: , DOI:
  43. Wojciechowski CL, Cardia JP, and Kantrowitz ER. 2002. Alkaline phosphatase from the hyperthermophilic bacterium T. maritima requires cobalt for activity Protein Sci. 4, PubMedID: 11910033, DOI:
  44. Camarero, J.A., et al. 2002. Autoregulation of a bacterial sigma factor explored by using segmental isotopic labeling and NMR Proc. Natl. Acad. Sci. USA. 99, PubMedID: 12084914, DOI:
  45. Wojciak, J.M., et al. 2002. Arm-site binding by lambda -integrase: solution structure and functional characterization of its amino-terminal domain Proc. Natl. Acad. Sci. USA . 99, PubMedID: 11904406, DOI:
  46. Moolenaar GF, et al. 2002. Cho, a second endonuclease involved in Escherichia coli nucleotide excision repair Proc. Natl. Acad. Sci. US. 99, PubMedID: 11818552, DOI:
  47. Cotton, G.J. and Muir, T.W. 2000. Generation of a dual-labeled fluorescence biosensor for Crk-II phosphorylation using solid-phase expressed protein ligation Chem. Biol. 4, PubMedID: 10780925, DOI:
  48. Zhang, Z., et al. 2003. The role of C-terminal tyrosine phosphorylation in the regulation of SHP-1 explored via expressed protein ligation J. Biol. Chem.. 278, PubMedID: 12468540, DOI:
  49. Scheibner, K.A., Zhang, Z. and Cole, P.A. 2003. Merging fluorescence resonance energy transfer and expressed protein ligation to analyze protein-protein interactions Anal. Biochem. . 317, PubMedID: 12758261, DOI:
  50. Bonsager, B.C., et al. 2003. Purification and characterization of the beta-trefoil fold protein barley alpha-amylase/subtilisin inhibitor overexpressed in Escherichia coli Protein Expr. Purif. . 2, PubMedID: 12880767, DOI:
  51. Wuebbens, M. M. and Rajagopalan, K. V. 2003. Mechanistic and mutational studies of Escherichia coli molybdopterin synthase clarify the final step of molybdopterin biosynthesis J. Biol. Chem. . 278, PubMedID: 12571226, DOI:
  52. Rak, A., et al. 2003. Structure of Rab GDP-dissociation inhibitor in complex with prenylated YPT1 GTPase Science. 302, PubMedID: 14576435, DOI:
  53. Yee, C.S., et al. 2003. Generation of the R2 subunit of ribonucleotide reductase by intein chemistry: insertion of 3-nitrotyrosine at residue 356 as a probe of the radical initiation process Biochemistry. 42, PubMedID: 14661967, DOI:
  54. Hong, S.H., and Maret, W. 2003. A fluorescence resonance energy transfer sensor for the beta-domain of metallothionein Proc. Natl. Acad. Sci. USA. 100, PubMedID: 12618543, DOI:
  55. Diao, H., et. al. 2007. Intein-mediated expression is an effective approach in the study of beta-defensins Biochem Biophys Res Commun. 357, PubMedID: 17445764, DOI:
  56. Kochinyan, S., et al. 2007. Use of intein-mediated phosphoprotein arrays to study substrate specificity of protein phosphatases Biotechniques. 42, PubMedID: 17269486 , DOI:
  57. Karow, A., et al. 2007. Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domains FEBS J. . 2, PubMedID: 17229151, DOI:
  58. Hassiepen, U., et. al. 2007. A sensitive fluorescence intensity assay for deubiquitinating proteases using ubiquitin-rhodamine110-glycine as substrate Anal Biochem.. 371, PubMedID: 17869210, DOI:
  59. Sun, L., et al. 2007. Design, preparation and use of ligated phosphoproteins: A novel approach to study protein phosphatases by dot blot array, ELISA and Western blot assays Methods. 42, PubMedID: 17532508, DOI:
  60. Hondal, R. J. 2009. Using chemical approaches to study selenoproteins-Focus on thioredoxin reductases Biochim. Biophys. Acta.. 1790, PubMedID: 19406205, DOI:
  61. Chattopadhaya, S., et al. 2009. Use of intein-mediated protein ligation strategies for the fabrication of functional protein arrays Methods Enzymol.. 462, PubMedID: 19632476, DOI:
  62. Chattopadhaya, S., et al. 2009. Site-specific covalent labeling of proteins inside live cells using small molecule probes Bioorg. Med. Chem.. 17, PubMedID: 18261914, DOI:
  63. Singleton, S. F., et al. 2002. Intein-mediated affinity-fusion purification of the Escherichia coliRecA protein Protein Expr. Purif. . 26, PubMedID: , DOI:
  64. Humphries, H. E., Christodoulides, M. and Heckels, J. E. 2002. Expression of the class 1 outer-membrane protein of Neisseria meningitidis in Escherichia coli and purification using a self-cleavable affinity tag Protein Expr. Purif. . 26, PubMedID: , DOI:
  65. Dorsch, S., et al. 2001. The VP1-unique region of parvovirus B19: amino acid variability and antigenic stability J. Gen. Virol. . 82 (pt 1) , PubMedID: 11125172, DOI:
  66. Wu, J.W., et al. 2001. Crystal structure of a phosphorylated Smad2. Recognition of phosphoserine by the MH2 domain and insights on Smad function in TGF-beta signaling Mol Cell.. 8, PubMedID: 11779503, DOI:
  67. Hanzawa, H., et al. 2001. In vitro assembly of phytochrome B apoprotein with synthetic analogs of the phytochrome chromophore Proc. Natl. Acad. Sci. USA . 98 , PubMedID: 11248126, DOI:
  68. Zuberek, J., et al. 2003. Phosphorylation of eIF4E attenuates its interaction with mRNA 5' cap analogs by electrostatic repulsion: intein-mediated protein ligation strategy to obtain phosphorylated protein RNA. 9, PubMedID: 12554876, DOI:
  69. David, R., et al. 2003. Semisynthesis and application of carboxyfluorescein-labelled biologically active human interleukin-8 Biol. Chem.. 384, PubMedID: 14719805, DOI:
  70. Sinars, C.R., et al. 2003. Structure of the large FK506-binding protein FKBP51, an Hsp90-binding protein and a component of steroid receptor complexes Proc. Natl. Acad. Sci. USA. 100, PubMedID: 12538866, DOI:
  71. Salazar, J.C., et al. 2003. Coevolution of an aminoacyl-tRNA synthetase with its tRNA substrates Proc. Natl. Acad. Sci. USA. 100, PubMedID: 14615592, DOI:
  72. Schaffer, L., et al. 2003. Assembly of high-affinity insulin receptor agonists and antagonists from peptide building blocks Proc. Natl. Acad. Sci. USA. 100, PubMedID: 12684539, DOI:
  73. Walters, K.J., et al. 2003. DNA -repair protein hHR23a alters its protein structure upon binding proteasomal subunit S5a Proc. Natl. Acad. Sci. USA . 100, PubMedID: , DOI:
  74. Bhat, R. K. and Berger, S. 2007. New and easy strategy for cloning, expression, purification, and characterization of the 5S subunit of transcarboxylase from Propionibacterium f. shermanii Prep. Biochem. Biotechnol.. 37, PubMedID: 17134979, DOI:
  75. Paulick, M. G., et al. 2007. Synthetic analogues of glycosylphosphatidylinositol-anchored proteins and their behavior in supported lipid bilayers J. Am. Chem. Soc. . 129, PubMedID: 17715922, DOI:
  76. Stasser, J. P., et. al. 2007. A multinuclear copper(I) cluster forms the dimerization interface in copper-loaded human copper chaperone for superoxide dismutase Biochem. . 46, PubMedID: 17902702, DOI:
  77. Hauser, P. S., et al. 2007. Expressed protein ligation using an N-terminal cysteine containing fragment generated in vivo from a pelB fusion protein Protein Expr. Purif.. 54, PubMedID: 17493830, DOI:
  78. Che, N., et. al. 2009. Soluble expression and one-step purification of a neurotoxin Huwentoxin-I in Escherichia coli Protein Expr Purif.. 65, PubMedID: 19217942, DOI:
Schematic Illustration of the IMPACT™ System
Expression and Purification of E. coli Maltose-Binding Protein (MBP) Using the pTXB1 Vector (pMXB10)

Lane 1: uninduced cell extract.
Lane 2: induced cell extract showing expressed fusion protein.
Lane 3: MBP fractions eluted after inducing cleavage overnight at 4°C.
Lane 4: MBP ligated to a peptide containing an N-terminal cysteine. Marker M is the Protein Ladder (NEB #P7703).

T7-Controlled Expression of a Non-Toxic Protein in E. coli Hosts
A T7 expression plasmid containing a gene encoding E. coli UDG was transformed into each host, grown to 0.6 OD and induced for 3 hours. Comparison of soluble extracts from uninduced (-) and induced (+) cells shows superior control of expression in the T7 Express hosts while maintaining high levels of induced expression.
Western Analysis of 6-His-tagged Brugia malayi Protein
A) B. malayi protein expressed at 20°C in BL21(DE3).
B) Soluble fractions of B. malayi protein expressed at 30°C in Lemo21(DE3).
Overnight Expression of a Membrane Protein - PhoA fusion
Lemo System™ enables simple, rapid optimization of membrane protein expression.
Disulfide Bond Formation
Disulfide bond formation in the cytoplasm of wild type E. coli is not favorable, while SHuffle is capable of correctly folding proteins with multiple disulfide bonds in the cytoplasm.
PfCHT1 Chitinase Activity Assayed from Crude Lysates
Plasmodium falciparum chitinase (PfCHT1) with three cysteines was expressed from a plasmid under the regulation of T7 promoter. After induction, cells were harvested and crude cell lysates were prepared. PfCHT1 was assayed using a chromogenic substrate (CalBioChem #474550) and standardized to protein concentration using Bradford reagent.
Improved Purity of His-Tagged Proteins with NiCo21(DE3)
Expression of Glutamyl tRNA Synthetase (6-His) in NiCo21(DE3) Competent E. coli followed by Ni-NTA purification. Eluent (E) from a Ni-NTA column was passed over a chitin column. The protein of interest elutes in the flow through (FT), while the CBD-tagged metal binding proteins remain bound (B) to the chitin resin ( NEB #S6651S). Purifications were performed according to manufacturers' recommended conditions. B) Contaminants ArnA, SlyD and Can are confirmed by Western blot using Anti-CBD Antibody (NEB #E8034S)
NiCo21(DE3) Two-Step Purification
Purification workflow of target protein that has been expressed in the NiCo21(DE3) strain of E. coli.
Optimization of YidC-GFP Expression with Lemo21(DE3)
Lemo21(DE3) achieves a higher level of expression than BL21(DE3) pLysS.
Lemo21(BE3) vs. BL21(DE3)

Protein expression with Lemo21(DE3) is very similar to BL21(DE3), with only a few minor changes.

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This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.