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


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.
Sewekow
Eva
E
Institute of Anatomy, Medical Faculty, Otto-von-Guericke University Magdeburg, Leipziger Str, 44, 39120 Magdeburg, Germany. eva.sewekow@med.ovgu.de
Kessler
Lars Christian
LC
Seidel-Morgenstern
Andreas
A
Rothkötter
Hermann-Josef
HJ
D000485:Allergens / Q000302:isolation & purification*; D052179:Antigens, Plant; D001688:Biological Products / Q000302:isolation & purification; D005591:Chemical Fractionation; D002852:Chromatography, Ion Exchange / Q000379:methods*; D018548:Cotyledon / Q000166:cytology; D003594:Cytoplasmic Granules / Q000737:chemistry*; D057927:Hydrophobic and Hydrophilic Interactions; D014674:Plant Proteins, Dietary / Q000302:isolation & purification; D030262:Soybean Proteins / Q000302:isolation & purification*; D013025:Glycine max / Q000166:cytology
D000485:Allergens; D052179:Antigens, Plant; D001688:Biological Products; D014674:Plant Proteins, Dietary; D030262:Soybean Proteins; C075135:P34 protein, Glycine max