Skip to main content
SpringerLink
Log in

Cation affinity purification of histidine-tagged proteins

  • Methods and Protocols
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract  

Protein purification is a basic technology in both biological research and industrial production, and efficient, convenient, economical, and environmentally friendly purification methods have always been pursued. In this study, it was found that alkaline earth metal cations (Mg2+, Ca2+) and alkali metal cations (Li+, Na+, K+) and even nonmetal cations (e.g., NH4+, imidazole, guanidine, arginine, lysine) can precipitate multi-histidine-tagged proteins (at least two tags in a whole protein) at low salts concentrations that are 1–3 orders of magnitude lower than salting-out, and precipitated proteins could be dissolved at moderate concentration of corresponding cation. Based on this finding, a novel cation affinity purification method was developed, which requires only three centrifugal separations to obtain highly purified protein with purification fold similar to that of immobilized metal affinity chromatography. The study also provides a possible explanation for unexpected protein precipitation and reminds researchers to consider the influence of cations on the experimental results. The interaction between histidine-tagged proteins and cations may also have broad application prospects.

Key points

• Histidine-tagged proteins can be precipitated by low-concentrations common cations

• A novel nonchromatographic protein purification method was developed

• Purified protein can be obtained in pellet form by only three centrifugations

Graphical abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

References 

  • Ansorge MB, Kula M (2000) Production of recombinant L-leucine dehydrogenase from Bacillus cereus in pilot scale using the runaway replication system E. coli[pIET98]. Biotechnol Bioeng 68:557–562

    Article  CAS  PubMed  Google Scholar 

  • Arakawa T, Timasheff SN (1984) Mechanism of protein salting in and salting out by divalent cation salts: balance between hydration and salt binding. Biochemistry 23:5912–5923

    Article  CAS  PubMed  Google Scholar 

  • Baird GS, Zacharias DA, Tsien RY (2000) Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci U S A 97:11984–11989

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Baldwin RL (1996) How Hofmeister ion interactions affect protein stability. Biophys J 71:2056–2063

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Blanco F, Lloyd DG, Alkorta I, Elguero J (2014) Neutral alkaline-metal and alkaline-earth-metal derivatives of imidazole and benzimidazole. J Phys Chem A 118:4195–4204

    Article  CAS  PubMed  Google Scholar 

  • Borrebaeck CA, Lönnerdal B, Etzler ME (1981) Metal chelate affinity chromatography of the Dolichos biflorus seed lectin and its subunits. FEBS Lett 130:194–196

    Article  CAS  Google Scholar 

  • Carlsson J, Mosbach K, Bülow L (1996) Affinity precipitation and site-specific immobilization of proteins carrying polyhistidine tails. Biotechnol Bioeng 51:221–228

    Article  PubMed  Google Scholar 

  • Daniele PG, Foti C, Gianguzza A, Prenesti E, Sammartano S (2008) Weak alkali and alkaline earth metal complexes of low molecular weight ligands in aqueous solution. Coord Chem Rev 252:1093–1107

    Article  CAS  Google Scholar 

  • Emmanuel AM, Ronald KC, Franois D (2003) Interactions with aromatic rings in chemical and biological recognition. Angew Chem Int Ed 42:1210–1250

    Article  Google Scholar 

  • Gallivan JP, Dougherty DA (1999) Cation-π interactions in structural biology. Proc Natl Acad Sci U S A 96:9459–9464

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hamilton S, Odili J, Pacifico MD, Wilson GD, Kupsch J (2003) Effect of imidazole on the solubility of a his-tagged antibody fragment. Hybrid Hybridomics 22:347–355

    Article  CAS  PubMed  Google Scholar 

  • Hefti MH, Vugt-Van V, der Toorn CJ, Dixon R, Vervoort J (2001) A novel purification method for histidine-tagged proteins containing a thrombin cleavage site. Anal Biochem 295:180–185

    Article  CAS  PubMed  Google Scholar 

  • Heyda J, Mason PE, Jungwirth P (2010) Attractive interactions between side chains of histidine-histidine and histidine-arginine-based cationic dipeptides in water. J Phys Chem B 114:8744–8749

    Article  CAS  PubMed  Google Scholar 

  • Ho JG, Middelberg AP, Ramage P, Kocher HP (2003) The likelihood of aggregation during protein renaturation can be assessed using the second virial coefficient. Protein Sci 12:708–716

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hochuli E, Bannwarth W, Dobeli H, Gentz R, Stuber D (1988) Genetic approach to facilitate purification of recombinant proteins with a novel metal chelate adsorbent. Nat Biotechnol 6:1321–1325

    Article  CAS  Google Scholar 

  • Joseph E, Singhvi G (2019) Multifunctional nanocrystals for cancer therapy: a potential nanocarrier. In: Grumezescu AM (ed) Nanomaterials for Drug Delivery and Therapy. Elesvier, Cambridge, pp 91–116

    Chapter  Google Scholar 

  • King TP (1972) Separation of proteins by ammonium sulfate gradient solubilization. Biochemistry 11:367–371

    Article  CAS  PubMed  Google Scholar 

  • Larsson PO, Mosbach K (1979) Affinity precipitation of enzymes. FEBS Lett 98:333–338

    Article  CAS  PubMed  Google Scholar 

  • Liao SM, Du QS, Meng JZ, Pang ZW, Huang RB (2013) The multiple roles of histidine in protein interactions. Chem Cent J 7:1–12

    Article  Google Scholar 

  • Lilius G, Persson M, Bülow L, Mosbach K (1991) Metal affinity precipitation of proteins carrying genetically attached polyhistidine affinity tails. Eur J Biochem 198:499–504

    Article  CAS  PubMed  Google Scholar 

  • McPherson DT, Morrow C, Minehan DS, Wu J, Hunter E, Urry DW (1992) Production and purification of a recombinant elastomeric polypeptide, G- (VPGVG)19-VPGV, from Escherichia coli. Biotechnol Prog 8:347–352

    Article  CAS  PubMed  Google Scholar 

  • Melnick J, Lis E, Park JH, Kinsland C, Mori H, Baba T, Perkins J, Schyns G, Vassieva O, Osterman A, Begley TP (2004) Identification of the two missing bacterial genes involved in thiamine salvage: thiamine pyrophosphokinase and thiamine kinase. J Bacteriol 186:3660–3662

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Meyer DE, Chilkoti A (1999) Purification of recombinant proteins by fusion with thermally-responsive polypeptides. Nat Biotechnol 17:1112–1115

    Article  CAS  PubMed  Google Scholar 

  • Misono H, Maeda H, Tuda K, Ueshima S, Miyazaki N, Nagata S (2005) Characterization of an inducible phenylserine aldolase from Pseudomonas putida 24–1. Appl Environ Microbiol 71:4602–4609

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Müller-Dethlefs K, Hobza P (2000) Noncovalent interactions: a challenge for experiment and theory. Chem Rev 100:143–168

    Article  PubMed  Google Scholar 

  • Piatkevich KD, Efremenko EN, Verkhusha VV, Varfolomeev SD (2010) Red fluorescent proteins and their properties. Russ Chem Rev 79:243–258

    Article  CAS  Google Scholar 

  • Pieters BJGE, van Eldijk MB, Nolte RJM, Mecinović J (2016) Natural supramolecular protein assemblies. Chem Soc Rev 45:24–39

    Article  CAS  PubMed  Google Scholar 

  • Porath J, Carlsson J, Olsson I, Belfrage G (1975) Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 258:598–599

    Article  CAS  PubMed  Google Scholar 

  • Riguero V, Clifford R, Dawley M, Dickson M, Gastfriend B, Thompson C, Wang SC, O’Connor E (2020) Immobilized metal affinity chromatography optimization for poly-histidine tagged proteins. J Chromatogr A 1629:461505

    Article  CAS  PubMed  Google Scholar 

  • Robichon C, Luo J, Causey TB, Benner JS, Samuelson JC (2011) Engineering Escherichia coli BL21 (DE3) derivative strains to minimize E. coli protein contamination after purification by immobilized metal affinity chromatography. Appl Environ Microbiol 77:4634–4646

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Salonen LM, Ellermann M, Diederich F (2011) Aromatic rings in chemical and biological recognition: energetics and structures. Angew Chem Int Ed 50:4808–4842

    Article  CAS  Google Scholar 

  • Samimi S, Maghsoudnia N, Eftekhari RB, Dorkoosh F (2019) Lipid-based nanoparticles for drug delivery systems. In: Mohapatra SS, Ranjan S, Dasgupta N, Mishra RK, Thomas S (eds) Characterization and Biology of Nanomaterials for Drug Delivery. Elsevier, Cambridge, pp 47–76

    Chapter  Google Scholar 

  • Schirwitz K, Schmidt A, Lamzin VS (2007) High-resolution structures of formate dehydrogenase from Candida boidinii. Protein Sci 16:1146–1156

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schneider HJ (2009) Binding mechanisms in supramolecular complexes. Angew Chem Int Ed 48:3924–3977

    Article  CAS  Google Scholar 

  • Strack RL, Strongin DE, Bhattacharyya D, Tao W, Berman A, Broxmeyer HE, Keenan RJ, Glick BS (2008) A noncytotoxic DsRed variant for whole-cell labeling. Nat Methods 5:955–957

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Swartz AR, Sun Q, Chen W (2017) Ligand-induced cross-linking of Z-elastin-like polypeptide-functionalized E2 protein nanoparticles for enhanced affinity precipitation of antibodies. Biomacromol 18:1654–1659

    Article  CAS  Google Scholar 

  • Timasheff SN, Arakawa T (1988) Mechanism of protein precipitation and stabilization by co-solvents. J Cryst Growth 90:39–46

    Article  CAS  Google Scholar 

  • Wingfield P (1998) Protein precipitation using ammonium sulfate. In: Current protocols in protein science. John Wiley & Sons, Inc., New Jersey, pp A.3F.1-A.3F.8

  • Wood DW (2014) New trends and affinity tag designs for recombinant protein purification. Curr Opin Struct Biol 26:54–61

    Article  CAS  PubMed  Google Scholar 

  • Young CL, Britton ZT, Robinson AS (2012) Recombinant protein expression and purification: a comprehensive review of affinity tags and microbial applications. Biotechnol J 7:620–634

    Article  CAS  PubMed  Google Scholar 

Download references

Funding

This work was financially supported by the National Natural Science Foundation of China (22078019).

Author information

Authors and Affiliations

  1. Department of Biological Science and Engineering, School of Chemistry and Biological Engineering, University of Science and Technology, Beijing, Beijing, 100083, China

    Hongxu Sun, Hongrui Wang, Qiwei Chen, Wenge Dong, Chao Gao, Hao Wu, Liangyu Hou & Hui Luo

  2. Department of Environmental Science and Engineering, School of Energy and Environmental Engineering, University of Science and Technology, Beijing, Beijing, 100083, China

    Haiyan Song, Hui Peng, Ren Li & Yanhong Chang

  3. Beijing Key Laboratory of Resource-Oriented Treatment of Industrial Pollutants, Beijing, 100083, China

    Haiyan Song, Hui Peng, Ren Li & Yanhong Chang

Authors
  1. Hongxu Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hongrui Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Qiwei Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wenge Dong
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Chao Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Haiyan Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hui Peng
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ren Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Hao Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Liangyu Hou
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Yanhong Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Hui Luo
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Conceptualization: HS and HL; formal analysis: HS and HW; funding acquisition: HL; investigation: HS, HW, QC, WD, CG, HS, HP, RL, HW, and LH; methodology: HS, HW, and HL; project administration: HL; supervision: YC and HL; validation: HS and HW; visualization: HS and HW; writing—original draft: HS; writing—review and editing: HS, YC, and HL.

Corresponding authors

Correspondence to Yanhong Chang or Hui Luo.

Ethics declarations

Ethics approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest

A patent application related to this work has been filed, in which HL, YC, HS, HW, and QC are named inventors. The other authors declare no conflict of interest.

Additional information

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 699 KB)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sun, H., Wang, H., Chen, Q. et al. Cation affinity purification of histidine-tagged proteins. Appl Microbiol Biotechnol 107, 2639–2651 (2023). https://doi.org/10.1007/s00253-023-12425-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-023-12425-3

Keywords

Search

Navigation