Skip to main content
SpringerLink
Log in

Per Aqueous Liquid Chromatography (PALC) as a Simple Method for Native Separation of Protein A

  • Original
  • Published:
Chromatographia Aims and scope Submit manuscript

A Correction to this article was published on 04 November 2017

This article has been updated

Abstract

Staphylococcal protein A (protein A) is an important protein frequently used in research studies within the fields of biomedicine and biotechnology. Due to some limitations in available protein purification methods which can hold the native structure of the protein A without changing the folding or adding histidine to structure of this protein, its separation in the native form is difficult. In this study, a new cost-effective and powerful technique was introduced for separation of the full-length and truncated forms of recombinant protein A, without any alteration in their 3D structures. Per aqueous liquid chromatography with bare silica gel stationary phase and water:acetonitrile as the mobile phase was proved to be an attractive choice among the range of separation methods. Similar to hydrophilic liquid chromatography, this method employs high percentage of water in mobile phase. The effects of mobile phase composition, pH, and salt concentration on the retention behavior of protein A on bare silica gel stationary phases were investigated. In this method, applying high amounts of aqueous solvent accompanied by a minimum percentage of organic solvent could successfully separate protein A with preservation of folding, and any affinity-tagged group such as histidine has not occurred on its structure. Purity of the fractions obtained by the proposed method was confirmed using SDS-PAGE, western blotting, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. According to the results of ELISA, separated proteins retained their ability of binding to antibody.

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

Change history

  • 04 November 2017

    Please correct the sequence of the authors to read as follows:

Abbreviations

PALC:

Per aqueous liquid chromatography

Protein A:

Staphylococcal protein A

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

MALDI-TOF MS:

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry

Fc:

Fragment crystallizable

IgG:

Immunoglobulin G

IMAC:

Immobilized metal ion affinity chromatography

HILIC:

Hydrophilic interaction liquid chromatography

ACN:

Acetonitrile

TFA:

Trifluoroacetic acid

BSA:

Bovine serum albumin

PBS:

Phosphate buffer saline

References

  1. Graille M, Stura EA, Corper AL, Sutton BJ, Taussig MJ, Charbonnier J-B, Silverman GJ (2000) PNAS 97:5399–5404

    Article  CAS  Google Scholar 

  2. Jungbauer A, Hahn R (2004) Curr Opin Drug Discov Devel 7:248–256

    CAS  Google Scholar 

  3. Lawman M, Thurmond M, Reis K, Gauntlett D, Boyle M (1984) Vet Immunol Immunopathol 6:291–305

    Article  CAS  Google Scholar 

  4. Dossett JH, Kronvall G, Williams RC, Quie PG (1969) J Immunol 103:1405–1410

    CAS  Google Scholar 

  5. Balint Jr JP (1991) Purification of protein a by affinity chromatography followed by anion exchange. US5075423

  6. Torres AR, Runkis WH (1999) Simple, environmentally benign, method for purifying protein A. WO1999010370A1

  7. Forsgren A, Sjöquist J (1966) J Immunol 97:822–827

    CAS  Google Scholar 

  8. Surade S (2007) Structural genomics on prokaryotic membrane proteins. Johann Wolfgang Goethe-University Frankfurt am Main, Germany

    Google Scholar 

  9. Wu J, FUutowiczK M (1999) Acta Biochim Pol 46:591–599

    CAS  Google Scholar 

  10. Schmitt J, Hess H, Stunnenberg HG (1993) Mol Biol Rep 18:223–230

    Article  CAS  Google Scholar 

  11. Bornhorst JA, Falke JJ (2000) Methods Enzymol 326:245–254

    Article  CAS  Google Scholar 

  12. Louwrier A (1999) Biotechnol Tech 13:329–330

    Article  CAS  Google Scholar 

  13. Carroll J, Fearnley IM, Walker JE (2006) PNAS 103:16170–16175

    Article  CAS  Google Scholar 

  14. Pedrali A, Tengattini S, Marrubini G, Bavaro T, Hemström P, Massolini G, Terreni M, Temporini C (2014) Molecules 19:9070–9088

    Article  Google Scholar 

  15. Tetaz T, Detzner S, Friedlein A, Molitor B, Mary J-L (2011) J Chromatogr A 1218:5892–5896

    Article  CAS  Google Scholar 

  16. Guo Y, Gaiki S (2005) J Chromatogr A 1074:71–80

    Article  CAS  Google Scholar 

  17. Knox J, Pryde A (1975) J Chromatogr A 112:171–188

    Article  CAS  Google Scholar 

  18. Periat A, Kohler I, Bugey A, Bieri S, Versace F, Staub C, Guillarme D (2014) J Chromatogr A 1356:211–220

    Article  CAS  Google Scholar 

  19. dos Santos Pereira A, David F, Vanhoenacker G, Sandra P (2009) J Sep Sci 32:2001–2007

    Article  Google Scholar 

  20. Gritti F, dos Santos Pereira A, Sandra P, Guiochon G (2010) J Chromatogr A 1217:683–688

    Article  CAS  Google Scholar 

  21. Ghaedmohammadi S, Rigi G, Zadmard R, Ricca E, Ahmadian G (2015) Mol Biotech 57:756–766

    Article  CAS  Google Scholar 

  22. Rigi G, Bahrami T, Armand R, Piruzeh Z (2016) Iranian J Health Sci 4:35–44

    Article  Google Scholar 

  23. Rigi G, Beyranvand P, Ghaedmohammadi S, Heidarpanah S, Noghabi KA, Ahmadian G (2015) J Phram Sci 104:6–11

  24. Rigi G, Mohammadi SG, Arjomand MR, Ahmadian G, Noghabi KA (2014) Biotechnol Appl Biochem 61:217–225

    Article  CAS  Google Scholar 

  25. Laemmli U (1970) Nature 227:680–685

    Article  CAS  Google Scholar 

  26. Gromova I, Celis JE (2006) Cell biol 4:421–429

    Google Scholar 

  27. Nielsen UB, Geierstanger BH (2004) J Immunol Methods 290:107–120

    Article  CAS  Google Scholar 

  28. Hobbs H, Reddy D, Rajeshwari R, Reddy A (1987) Plant Dis 71:747

    Article  Google Scholar 

  29. Parker J, Guo D, Hodges R (1986) Biochem 25:5425–5432

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors would like to thank the National Institute of Genetic Engineering and Biotechnology (NIGEB) of Iran for providing the necessary equipment.

Author information

Author notes

  1. Garshasb Rigi and Mohsen Farhadpour are joint first authors.

Authors and Affiliations

  1. Pharmaceutical and Medicinal Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Center, Dokki, Cairo, 12622, Egypt

    Hassan Y. Aboul-Enein

  2. Department of Genetics, Faculty of Basic Sciences, University of Shahrekord, Shahrekord, Iran

    Garshasb Rigi & Alireza Ghasempour

  3. Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G.C., Evin, Tehran, Iran

    Mohsen Farhadpour & Alireza Ghasempour

  4. Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), P.O. Box 14155-6343, Tehran, Iran

    Gholamreza Ahmadian

  5. Department of Molecular Genetics, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran

    Gholamreza Ahmadian

Authors
  1. Hassan Y. Aboul-Enein
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Garshasb Rigi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mohsen Farhadpour
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alireza Ghasempour
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Gholamreza Ahmadian
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Hassan Y. Aboul-Enein, Alireza Ghasempour or Gholamreza Ahmadian.

Ethics declarations

Conflict of interest

The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript, apart from those disclosed. No writing assistance was utilized in the production of this manuscript. The authors declare no conflicts of interest.

Ethical approval

All applicable international, national, and institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants or animals performed by any of the authors.

Additional information

A correction to this article is available online at https://doi.org/10.1007/s10337-017-3432-x.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 33 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Aboul-Enein, H.Y., Rigi, G., Farhadpour, M. et al. Per Aqueous Liquid Chromatography (PALC) as a Simple Method for Native Separation of Protein A. Chromatographia 80, 1633–1639 (2017). https://doi.org/10.1007/s10337-017-3412-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10337-017-3412-1

Keywords

Search

Navigation