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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 30K 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 one-step 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.
Conclusions
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.
© 2008 Sewekow et al; licensee BioMed Central Ltd.
