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TPA Purification Protocols

Tissue plasminogen activator or TPA is a type of serine protease, which is protein able to destroy blood clots. TPA is found in the cells that internally cover the blood vessels. TPA protocols are part of the pharmaceutical and medical research on strokes, as well as heart and lung diseases, such as pulmonary embolism and myocardial infarction. TPA purification refers to the isolation of this protein from other molecules through a series of processes, including chromatography and electrophoresis.

  1. Purification of TPA from Hog Kidney

    • This protocol is used in studies of fibrinolysis, the process of breaking the product of coagulation called fibrin, related to the formation of tumors. Technicians extract TPA from acetone-dried hog kidney with potassium acetate buffer, at pH 4.2. Then, they add ammonium sulfate to induce cell precipitation. They add n-butyl-sepharose to the samples, which are then analyzed on a chromatographer. Concanavalin A is also added and chromatograph.y to separate different proteins is performed.

    Purification of TPA from Chinese Hamster Ovary and E. coli Cells

    • Protein purity is an important factor in the success of biological experiments. Purified TPA is also commercially available, thus saving time and avoiding the risk of jeopardizing the experiment due to poorly purified solution. This protocol involves the purification processes of TPA in mammal and bacterial cells. Technicians precipitate cell samples, filtrate them and perform affinity and gel chromatography analysis. Later, they analyze and compare the results, prior to commercial production.

    Expression of Active Human TPA in E. coli

    • Clarified cell lysates are added to a lysine--Sepharose column, analyzed in a chromatographer and washed with a solution of sodium chlorine. Another sample is loaded onto a second chromatography column containing proteins that binds to TPA with a very high affinity. A sample from the second column is mixed with a solution of zinc-sepharose. The resulting protein shows more than 90 percent purity, according to the journal "Applied and Environmental Microbiology."

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