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:: Volume 12, Number 2 (Scientific Journal of Kurdistan University of Medical Sciences 2007) ::
SJKU 2007, 12(2): 77-83 Back to browse issues page
Purification of Kunitz soybean trypsin inhibitor using affinity chromathography
mr Y Shakiba, dr A, Mostafaie , mr SH Parvaneh
Abstract:  

  Background and aim : soybean trypsin inhibitor, a single chain protein with 181 amino acids and two disulfide bonds, has special tendency for pancreatic trypsin enzyme. This protein exisits in soybean abundantly and showes high resistance to heating and chemicals. Various procedures such as combination of different chromatographic methods, immobilized metal affinity chromatography and preparative electrophoresis have been used to purify this protein.

  Materials and Methods : Pulverized soybean was defatted with methanol and the reminder was extracted after being solved in dionized water. Storage proteins in solution was eliminated by use of isoelelctric precipitation method. Trypsin inhibitor riched fraction was adsorbed on an affinity column with trypsin as the ligand. After washing unbonded proteins, trypsin inhibitor was eluted from the collumn by decreasing pH. Purity and activity of the inhibitor was assessed by SDS-PAGE and an spectrophotometric method, respectively.

  Results : SDS-PAGE demonstrated a protein with a single band and molecular weight of 20 KDa with purity of more than 99 percent under reducing and non-reducing conditions. Activity of the purified inhibitor protein was 3600 TIU/mg. The results showed that overloading of the affinity collumn to gether with chromatography at pH of 6 to 8 increased the purity.

Conclusion : The proposed method is simple and efficient for preparation of large amounts of pure trypsin inhibitor. In addition, this method does not need any further steps such as dialysis
Keywords: Key words: Soybean, Trypsin inhibitor, Trypsin, Affinity chromatography
Full-Text [DOC 3226 kb]      
Type of Study: Research | Subject: General
Received: 2009/04/8
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Volume 12, Number 2 (Scientific Journal of Kurdistan University of Medical Sciences 2007) Back to browse issues page
مجله علمی دانشگاه علوم پزشکی کردستان Scientific Journal of Kurdistan University of Medical Sciences
مجله علمی دانشگاه علوم پزشکی کردستان Scientific Journal of Kurdistan University of Medical Sciences
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