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  • 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. 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
    2. 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
    3. 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
    4. 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
    5. 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
    6. 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
    7. 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
    8. 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
    9. Zhong, Q., et al. (2002)Endosomal localization and function of sorting Proc. Natl. Acad. Sci. USA 99, 6767-6772.
    10. 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.
    11. Singleton, S. F., et al. (2002)Intein-mediated affinity-fusion purification of the Escherichia coliRecA protein Protein Expr. Purif.  26, 476-488.
    12. Nakano, S., et al. (2002)Multiple pathways of Spx (YjbD) proteolysis in Bacillus subtilis J. Bacteriol 184, 3664-3670. PubMedID: 12057962
    13. 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
    14. 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
    15. 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
    16. 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.
    17. 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
    18. 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
    19. 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
    20. 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
    21. 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
    22. David, R., et al. (2003)Semisynthesis and application of carboxyfluorescein-labelled biologically active human interleukin-8 Biol. Chem. 384, 1619-1630. PubMedID: 14719805
    23. Rak, A., et al. (2003)Structure of Rab GDP-dissociation inhibitor in complex with prenylated YPT1 GTPase Science 302, 646-50. PubMedID: 14576435
    24. 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
    25. 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
    26. 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
    27. 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
    28. 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.
    29. 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
    30. 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
    31. 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
    32. 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
    33. Durek, T., et al. (2004)Synthesis of fluorescently labeled mono- and diprenylated Rab7 GTPase J. Am. Chem. Soc. 126, 16368-16378. PubMedID: 15600338
    34. Lue, R.Y., et al. (2004)Versatile protein biotinylation strategies for potential high-throughput proteomics J Am. Chem. Soc. 126, 1055-1062. PubMedID: 14746473
    35. 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
    36. 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.
    37. 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.
    38. Ge, X., et. al. (2005)Self-cleavable stimulus responsive tags for protein purification without chromatography J. Am. Chem. Soc 127, 11228-9. PubMedID: 16089436
    39. 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
    40. 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
    41. Hackenberger, C. P., et. al. (2006)Expression of N-terminal Cys-protein fragments using an intein refolding strategy Bioorg. Med. Chem. 14, 5043-5048.
    42. Gottlieb, D., et al. (2006)Intein-mediated in vitro synthesis of lipidated Ras proteins Chem. Commun. (Camb) 21, 260-262. PubMedID: 16391727
    43. 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.
    44. 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
    45. 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
    46. Kalia, J., et al. (2006)Reactivity of intein thioesters: appending a functional group to a protein Chembiochem. 7, 1375-1383. PubMedID: 16897799
    47. Eckenroth, B., et al. (2006)Semisynthesis and characterization of mammalian thioredoxin reductase Biochem. 45, 5158-70. PubMedID: 16618105
    48. 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
    49. 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
    50. 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
    51. 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
    52. 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
    53. 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
    54. 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
    55. 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
    56. 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
    57. 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
    58. Kochinyan, S., et al. (2007)Use of intein-mediated phosphoprotein arrays to study substrate specificity of protein phosphatases Biotechniques 42, 63-69. PubMedID: 17269486
    59. Ottesen, J. et al. (2008)An amalgamation of solid phase peptide synthesis and ribosomal peptide synthesis Biopolymers 90, 406-414. PubMedID: 17636509
    60. 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
    61. 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
    62. 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
    63. 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
    64. Zhao, Z., et. al. (2008)Purification of green fluorescent protein using a two-intein system Appl. Microbiol. Biotechnol. 77, 1175-1180. PubMedID: 17973109
    65. 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
    66. 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
    67. 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
    68. Hondal, R. J. (2009)Using chemical approaches to study selenoproteins-Focus on thioredoxin reductases Biochim. Biophys. Acta. 1790, 1501-1512. PubMedID: 19406205
    69. 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
    70. Chen, J., et al. (2010)Chemically ubiquitylated PCNA as a probe for eukaryotic translesion DNA synthesis Nat. Chem. Biol. 6, 270-272. PubMedID: 20208521
    71. 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
    72. Lu, X., et al. (2010)Designed semisynthetic protein inhibitors of ub/ubl e1 activating enzymes J. Am. Chem. Soc. 132, 1748-1749. PubMedID: 20099854
    73. 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
    74. Manconi, B., et. al. (2010)Expression, purification, phosphorylation and characterization of recombinant human statherin Protein Expr Purif. 69, 219-225.
    75. Lu, M., et. al. (2010)Purification of untagged HIV-1 reverse transcriptase by affinity chromatography Protein Expr. Purif. 71, 231-239. PubMedID: 20060474

    Schematic Illustration of Purification Using pMAL™ System

    Protein Expression Using pMAL™

    SDS-polyacrylamide gel electrophoresis of fractions from the purification of MBP-paramyosin-ΔSal.
    Lane 1: Protein Ladder (NEB #P7703).
    Lane 2: uninduced cells.
    Lane 3: induced cells.
    Lane 4: purified protein eluted from amylose column with maltose.
    Lane 5: purified protein after Factor Xa cleavage.
    Lane 6: paramyosin fragment in flow-through from second amylose column.

    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.