Purification Of Porcine T-PA With Rationally Designed Peptide Ligand

Tissue Plasminogen Activator (t-PA) is a kind of valuable protein which is being widely used in thrombus therapy. In this paper, t-PA have been purified from porcine heart by affinity chromatography coupled with peptide ligand QDES which was rationally designed to t-PA.The carboxyl groups of QDES were used to couple the free amino groups of EAH Sepharose 4B with the carbodiimide coupling method, and immobilized density was 5μmol/ml. Adsorption between different proteins (α-amylase,lysozyme,insulin) and affinity sorbent was carried out, and the result was thatα-amylase and lysozyme can't adsorb the affinity sorbent, but insulin can get high affinity capability. Because of the adsorption region of insulin similar to t-PA, it is concluded that t-PA also has high affinity for the same sorbent.In order to value the result of affinity separation, the fresh porcine heart was prepared for acetone powder. After extrcation and salt out, the t-PA crude solution was got. The concentration of protein was measured with Bradford method, the activity of t-PA was was measured with fibrinolysis method. 159g was obtained from 1kg porcine heart; 35.12mg protein can be obtained from 100g acetone powder; the product has a. After affinity separation of t-PA crude solution, the purity of final product is 91%, the specific activity is 34037 IU/mg, and 1.3mg t-PA can be obtained from 1kg porcine heart. The adsorption of different proteins to affinity column has showed that the electrostatic interaction plays an important role in the affinity action, which is consistent with the result of rational design.Above all, the rationally designed affinity ligand can be used for separation t-PA from porcine heart successfully and efficiently.