Affinity Purification of Heme-Tagged Proteins

  • Wesley B. AsherEmail author
  • Kara L. Bren
Part of the Methods in Molecular Biology book series (MIMB, volume 1177)


Protein affinity purification techniques are widely used for isolating pure target proteins for biochemical and structural characterization. Herein, we describe the protocol for affinity-based purification of proteins expressed in Escherichia coli that uses the coordination of a peptide tag covalently modified with heme c, known as a heme-tag, to an l-histidine immobilized Sepharose resin. This approach provides an affinity purification tag visible to the eye, facilitating tracking of the protein. In addition, we describe methods for specifically detecting heme-tagged proteins in SDS-PAGE gels using a heme-staining procedure and for quantifying the proteins using a pyridine hemochrome assay.

Key words

Affinity protein purification Affinity tag Heme-tag Visible-tag l-histidine immobilized Sepharose chromatography Protein quantification Visible tracking 


  1. 1.
    Young CL, Britton ZT, Robinson AS (2012) Recombinant protein expression and purification: a comprehensive review of affinity tags and microbial applications. Biotechnol J 7:620–624PubMedCrossRefGoogle Scholar
  2. 2.
    Chelur D, Unal O, Scholtyssek M, Strickler J (2008) Fusion tags for protein expression and purification. BioPharm Int 21(Suppl 6):38–46Google Scholar
  3. 3.
    Arnau J, Lauritzen C, Petersen GE et al (2006) Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins. Protein Expr Purif 48:1–13PubMedCrossRefGoogle Scholar
  4. 4.
    Lichty JJ, Malecki JL, Agnew HD et al (2005) Comparison of affinity tags for protein purification. Protein Expr Purif 41:98–105PubMedCrossRefGoogle Scholar
  5. 5.
    Asher WB, Bren KL (2010) A heme fusion tag for protein affinity purification and quantification. Protein Sci 19:1830–1839PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Finn RD, Kapelioukh L, Paine MJI (2005) Rainbow tags: a visual tag system for recombinant protein expression and purification. Biotechniques 38:387–392PubMedCrossRefGoogle Scholar
  7. 7.
    Braun M, Rubio IG, Thöny-Meyer L (2005) A heme tag for in vivo synthesis of artificial cytochromes. Appl Microbiol Biotechnol 67:234–239PubMedCrossRefGoogle Scholar
  8. 8.
    Allen JWA, Ferguson SJ (2006) What is the substrate specificity of the system I cytochrome c biogenesis apparatus? Biochem Soc Trans 34:150–151PubMedCrossRefGoogle Scholar
  9. 9.
    Kranz RG, Richard-Fogal C, Taylor JS et al (2009) Cytochrome c biogenesis: mechanism for covalent modifications and trafficking of heme and for heme-iron redox control. Microbiol Mol Biol Rev 73:510–528PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Choi JH, Lee SY (2004) Secretory and extracellular production of recombinant proteins using Escherichia coli. Appl Microbiol Biotechnol 64:625–635PubMedCrossRefGoogle Scholar
  11. 11.
    Sletta H, Tøndervik A, Hakvåg S et al (2007) The presence of N-terminal secretion signal sequences leads to strong stimulation of the total expression levels of three medically important proteins during high-cell-density cultivations of Escherichia coli. Appl Environ Microbiol 73:906–912PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Canters GW (1987) The azurin gene from Pseudomonas aeruginosa codes for a pre-protein with a signal peptide. FEBS Lett 212:168–172PubMedCrossRefGoogle Scholar
  13. 13.
    Fee JA, Chen Y, Todaro TR et al (2000) Integrity of Thermus thermophilus cytochrome c(552) synthesized by Escherichia coli cells expressing the host-specific cytochrome c maturation genes, ccmABCDEFGH. Biochemical, spectral, and structural characterization of the recombinant protein. Protein Sci 9:2074–2084PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Arslan E, Schulz H, Zufferey R et al (1998) Overproduction of the Bradyrhizobium japonicum c-type cytochrome subunits of the cbb 3 oxidase in Escherichia coli. Biochem Biophys Res Commun 251:744–747PubMedCrossRefGoogle Scholar
  15. 15.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  16. 16.
    Francis RT, Becker RR (1984) Specific indication of hemoproteins in polyacrylamide gels using a double-staining process. Anal Biochem 136:509–514PubMedCrossRefGoogle Scholar
  17. 17.
    Berry EA, Trumpower BL (1987) Simultaneous determination of hemes-a, hemes-b, and hemes-c from pyridine hemochrome spectra. Anal Biochem 161:1–15PubMedCrossRefGoogle Scholar
  18. 18.
    Asher WB, Bren KL (2012) Cytochrome c heme lyase can mature a fusion peptide composed of the amino-terminal residues of horse cytochrome c. Chem Commun 48:8344–8346CrossRefGoogle Scholar
  19. 19.
    Petersen TN, Brunak S, Heijne G et al (2011) SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 8:785–786PubMedCrossRefGoogle Scholar
  20. 20.
    Smialowski P, Martin-Galiani AJ, Mikolajka A et al (2006) Protein solubility: sequence based prediction and experimental verification. Bioinformatics 23:2536–2542PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Division of Molecular Therapeutics Department of PsychiatryColumbia UniversityNew YorkUSA
  2. 2.Department of ChemistryUniversity of RochesterRochesterUSA

Personalised recommendations