Open Access Highly Accessed Methodology article

Isolation of soybean protein P34 from oil bodies using hydrophobic interaction chromatography

Eva Sewekow1, Lars Christian Kessler2, Andreas Seidel-Morgenstern23 and Hermann-Josef Rothkötter1*

Author Affiliations

1 Institute of Anatomy, Medical Faculty, Otto-von-Guericke University Magdeburg, Leipziger Str. 44, 39120 Magdeburg, Germany

2 Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstr.1, 39106 Magdeburg, Germany

3 Institute of Process Engineering, Otto von Guericke University, Universitätsplatz 2, D-39106 Magdeburg, Germany

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BMC Biotechnology 2008, 8:27  doi:10.1186/1472-6750-8-27

Published: 11 March 2008

Abstract

Background

Soybeans play a prominent role in allergologic research due to the high incidence of allergic reactions. For detailed studies on specific proteins it is necessary to have access to a large amount of pure substance.

Results

In this contribution, a method for purifying soybean (Glycine max) protein P34 (also called Gly m Bd 30 K or Gly m 1) using hydrophobic interaction chromatography is presented. After screening experiments using 1 mL HiTrap columns, Butyl Sepharose 4 FF was selected for further systematic investigations. With this stationary phase, suitable operation conditions for two-step gradient elution using ammonium sulphate were determined experimentally. The separation conditions obtained in a small column could be scaled up successfully to column volumes of 7.5 and 75 mL, allowing for high product purities of almost 100% with a yield of 27% for the chromatographic separation step. Conditions could be simplified further using a onestep gradient, which gave comparable purification in a shorter process time. The identity of the purified protein was verified using in-gel digestion and mass spectrometry as well as immunological techniques.

Conclusion

With the technique presented it is possible to produce, within a short timeframe, pure P34, suitable for further studies where an example antigen is needed.