Protein Expression

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.

E. coli includes these subcategories:

IMPACT™ Information
pMAL™ Information

    Publications related to E. coli:

  1. Lu, M., et. al. (2010). Purification of untagged HIV-1 reverse transcriptase by affinity chromatography Protein Expr. Purif.. 71, 231-239. PubMedID: 20060474
  2. Chen, J., et al. (2010). Chemically ubiquitylated PCNA as a probe for eukaryotic translesion DNA synthesis Nat. Chem. Biol. . 6, 270-272. PubMedID: 20208521
  3. 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
  4. Karukurichi, K. et al. (2010). Analysis of p300/CBP histone acetyltransferase regulation using circular permutation and semisynthesis J. Am. Chem. Soc. . 132, 1222- 1223. PubMedID: 20063892
  5. 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
  6. Manconi, B., et. al. (2010). Expression, purification, phosphorylation and characterization of recombinant human statherin Protein Expr Purif.. 69, 219-225.
  7. Lu, X., et al. (2010). Designed semisynthetic protein inhibitors of ub/ubl e1 activating enzymes J. Am. Chem. Soc. . 132, 1748-1749. PubMedID: 20099854
  8. Chattopadhaya, S., et al. (2009). Use of intein-mediated protein ligation strategies for the fabrication of functional protein arrays Methods Enzymol.. 462, 195-223. PubMedID: 19632476
  9. Hondal, R. J. (2009). Using chemical approaches to study selenoproteins-Focus on thioredoxin reductases Biochim. Biophys. Acta.. 1790, 1501-1512. PubMedID: 19406205
  10. Che, N., et. al. (2009). Soluble expression and one-step purification of a neurotoxin Huwentoxin-I in Escherichia coli Protein Expr Purif.. 65, 154-159. PubMedID: 19217942
  11. Chattopadhaya, S., et al. (2009). Site-specific covalent labeling of proteins inside live cells using small molecule probes Bioorg. Med. Chem.. 17, 981-9. PubMedID: 18261914
  12. Zhao, Z., et. al. (2008). Purification of green fluorescent protein using a two-intein system Appl. Microbiol. Biotechnol.. 77, 1175-1180. PubMedID: 17973109
  13. Ottesen, J. et al. (2008). An amalgamation of solid phase peptide synthesis and ribosomal peptide synthesis Biopolymers. 90, 406-414. PubMedID: 17636509
  14. 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, 736-747. PubMedID: 18305193
  15. Babu, S.K., et. al. (2008). Construction of intein mediated hGMCSF expression vector and its prufication in Pichia pastoris Protein Exp. Purif.. 57, 201-205. PubMedID: 18309571
  16. Chen, Y. Q., et. al. (2008). Expression of a cytotoxic cationic antibacterial peptide in Escherichia coli using two fusion partners Protein Exp. Purif.. 57, 303-311. PubMedID: 17977015
  17. 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, 151-164. PubMedID: 17936301
  18. 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, 13-26. PubMedID: 17134979
  19. Diao, H., et. al. (2007). Intein-mediated expression is an effective approach in the study of beta-defensins Biochem Biophys Res Commun. 357, 840-846. PubMedID: 17445764
  20. Kochinyan, S., et al. (2007). Use of intein-mediated phosphoprotein arrays to study substrate specificity of protein phosphatases Biotechniques. 42, 63-69. PubMedID: 17269486
  21. Paulick, M. G., et al. (2007). Synthetic analogues of glycosylphosphatidylinositol-anchored proteins and their behavior in supported lipid bilayers J. Am. Chem. Soc. . 129, 11543-11550. PubMedID: 17715922
  22. Karow, A., et al. (2007). Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domains FEBS J. . 2, 463-473. PubMedID: 17229151
  23. 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, 11845-11856. PubMedID: 17902702
  24. Hassiepen, U., et. al. (2007). A sensitive fluorescence intensity assay for deubiquitinating proteases using ubiquitin-rhodamine110-glycine as substrate Anal Biochem.. 371, 201-207. PubMedID: 17869210
  25. 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, 227-233. PubMedID: 17493830
  26. 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, 220-226. PubMedID: 17532508
  27. Demidov, V.V., and Broude, N.E. (2006). Profluorescent protein fragments for fast bimolecular fluorescence complementation in vitro Nat. Protoc.. 1, 714-719. PubMedID: 17406301
  28. Kalia, J., et al. (2006). Reactivity of intein thioesters: appending a functional group to a protein Chembiochem.. 7, 1375-1383. PubMedID: 16897799
  29. Gottlieb, D., et al. (2006). Intein-mediated in vitro synthesis of lipidated Ras proteins Chem. Commun. (Camb). 21, 260-262. PubMedID: 16391727
  30. 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, 343-347. PubMedID: 16712508
  31. 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, 465-472.
  32. 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, 6194-6202. PubMedID: 16407279
  33. Eckenroth, B., et al. (2006). Semisynthesis and characterization of mammalian thioredoxin reductase Biochem.. 45, 5158-70. PubMedID: 16618105
  34. Lee, N.Y., et al. (2006). Structure and dynamics of the epidermal growth factor receptor C-terminal phosphorylation domain Protein Sci.. 15, 1142-52. PubMedID: 16597832
  35. Hackenberger, C. P., et. al. (2006). Expression of N-terminal Cys-protein fragments using an intein refolding strategy Bioorg. Med. Chem.. 14, 5043-5048.
  36. 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, 152-163. PubMedID: 15721783
  37. Ge, X., et. al. (2005). Self-cleavable stimulus responsive tags for protein purification without chromatography J. Am. Chem. Soc. 127, 11228-9. PubMedID: 16089436
  38. Girish, A., et al. (2005). Site-specific immobilization of proteins in a microarray using intein-mediated protein splicing Bioorg. Med. Chem. Lett.. 15, 2447-51. PubMedID: 15863295
  39. Ingham, A.B., et al. (2005). A versatile system for the expression of nonmodified bacteriocins inEscherichia coli J. Appl. Microbiol. . 98, 676-683. PubMedID: 15715871
  40. Morassutti, C., et al. (2005). Expression of SMAP-29 cathelicidin-like peptide in bacterial cells by intein-mediated system Protein Expr. Purif.. 39, 160-168.
  41. 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 . 281-290.
  42. 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, 6397-6402. PubMedID: 15087498
  43. Hemelaar, J.et al. (2004). Specific and covalent targeting of conjugating and deconjugating enzymes of ubiquitin-like proteins Mol. Cell Biol.. 24, 84-95. PubMedID: 14673145
  44. 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), 2407-10. PubMedID: 15109622
  45. Lue, R.Y., et al. (2004). Versatile protein biotinylation strategies for potential high-throughput proteomics J Am. Chem. Soc.. 126, 1055-1062. PubMedID: 14746473
  46. Durek, T., et al. (2004). Synthesis of fluorescently labeled mono- and diprenylated Rab7 GTPase J. Am. Chem. Soc.. 126, 16368-16378. PubMedID: 15600338
  47. 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, 101-110. PubMedID: 14764935
  48. Pezza, J.A., Allen, K.N. and Tolan, D.R. (2004). Intein-mediated purification of a recombinantly expressed peptide Chem Commun (Camb). 21, 2412-2413. PubMedID: 15514791
  49. Guo, C., et al. (2004). Intein-mediated fusion expression, high efficient refolding, and one-step purification of gelonin toxin Protein Expr Purif.. 37, 361-367.
  50. Durek T., Goody R.S and Alexandrov K. (2004). In vitro semisynthesis and applications of C-terminally modified Rab proteins Methods Mol Biol.. 283, 233-244. PubMedID: 15197315
  51. 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, 52-61. PubMedID: 12554876
  52. 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, 226-232. PubMedID: 12758261
  53. 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, 14523-14532. PubMedID: 12571226
  54. 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, 868-873. PubMedID: 12538866
  55. 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, 4668-4674. PubMedID: 12468540
  56. Rak, A., et al. (2003). Structure of Rab GDP-dissociation inhibitor in complex with prenylated YPT1 GTPase Science. 302, 646-50. PubMedID: 14576435
  57. 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
  58. David, R., et al. (2003). Semisynthesis and application of carboxyfluorescein-labelled biologically active human interleukin-8 Biol. Chem.. 384, 1619-1630. PubMedID: 14719805
  59. Schaffer, L., et al. (2003). Assembly of high-affinity insulin receptor agonists and antagonists from peptide building blocks Proc. Natl. Acad. Sci. USA. 100, 4435-9. PubMedID: 12684539
  60. Salazar, J.C., et al. (2003). Coevolution of an aminoacyl-tRNA synthetase with its tRNA substrates Proc. Natl. Acad. Sci. USA. 100, 13863-13868. PubMedID: 14615592
  61. 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, 2255-2260. PubMedID: 12618543
  62. 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, 12694-12699.
  63. 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, 14541-14552. PubMedID: 14661967
  64. Nakano, S., et al. (2002). Multiple pathways of Spx (YjbD) proteolysis in Bacillus subtilis J. Bacteriol. 184, 3664-3670. PubMedID: 12057962
  65. Singleton, S. F., et al. (2002). Intein-mediated affinity-fusion purification of the Escherichia coliRecA protein Protein Expr. Purif. . 26, 476-488.
  66. Moolenaar GF, et al. (2002). Cho, a second endonuclease involved in Escherichia coli nucleotide excision repair Proc. Natl. Acad. Sci. US. 99, 1467-1472. PubMedID: 11818552
  67. Wojciechowski CL, Cardia JP, and Kantrowitz ER. (2002). Alkaline phosphatase from the hyperthermophilic bacterium T. maritima requires cobalt for activity Protein Sci. 4, 903-911. PubMedID: 11910033
  68. 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, 8536-8541. PubMedID: 12084914
  69. 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, 3434-3439. PubMedID: 11904406
  70. Zhong, Q., et al. (2002). Endosomal localization and function of sorting Proc. Natl. Acad. Sci. USA. 99, 6767-6772.
  71. 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, 243-8.
  72. Dorsch, S., et al. (2001). The VP1-unique region of parvovirus B19: amino acid variability and antigenic stability J. Gen. Virol. . 82 (pt 1), 191-199. PubMedID: 11125172
  73. 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, 1277-1289. PubMedID: 11779503
  74. 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, 3612-3617. PubMedID: 11248126
  75. 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, 253-261. PubMedID: 10780925

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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.