The Purification Of Kallikrein By Biomimic Affinity Chromatograph

Kallikreins are a subgroup of the serine protease family and are known to have diverse physiological functions. It has been found exits two classes of kallikrein in the body of human and animals. Plasma kallkrein and tissue kallikrein. Both of them can specifically cleaving the natural substrate, kininogen, at two points to release Lys-bradykinin which can bind with the B2 receptors to release the second messenger to cause vasodilatation, increased vascular permeability, pain and smooth muscle contraction, but it also initiates the intrinsic pathway of the blood coagulation cascade, resulting in fibrin clot formation. The specificity of tissue kallikreins is characterized by a marked preference for arginine over lysine residue in the substrate-binding pocket. Several clinical researchers argued that the decrease of kallikrein content in body is correspond with the symptom of hypertension and diabetes compared to the control, and the increase of kallikrein is parallel to the happen of asthma and arthritis.Because of having many pharmaceutical and physiological actions, kallikrein has been extensively used in clinic. The demand of kallikrein in recent market is increasing as the amplification of the population of diabetes. Most of the kallikreins used in clinic at present are porcine pancreatic kallikrein and urinary kallikrein is the minority. Although protokaryon and eukaryon expression systems had been constructed successfully, there still exist several shortcomings such as low yield or low enzyme activity, which limited them industrialization. So the porcine pancreatic kallikrein is the majority used in clinic than the genengineering product. And most of kallikreins used for drugs are predicated by ion-exchange chromatography, which needs at least 3 steps, and the yield is low. New techniques with less handle-steps and higher yield will more competitive than the conventional purification methods and more used in the future.Six kinds of ligands for the selective purification by affinity chromatography of porcine pancreatic kallikrein were designed according to the 3D structure of kallikrein and of its favorite substrate, Phe-Arg dipeptide. One of the ligands, trptamine immobilized to Sepharose CL-4B via triazine dyes, purified pancreatic kallikrein 290-fold in one step from a crude pancreatic acetone extract. The kallikrein was purified 1140 fold when the DEAE-FF-Sepharose was used to purify the elution of Tip column. The final specific activity of 1303KU/mg was obtained after two steps purification, and the yield was 60%.