Rapid and high-yield purification of porcine heart tissue-type plasminogen activator by heparin-sepharose choromatography.
A rapid and high-yield procedure for the purification of single polypeptide tissue-type plasminogen activator (t-PA) from porcine heart tissue has been developed. Delipidated heart tissue was extracted with 0.45 M potassium acetate. The extract was fractionated with ammonium sulfate and purified by a combination of affinity chromatography on heparin-Sepharose CL-6B and gel filtration on Toyopearl HW-55S. The final product had a specific activity of 220,000 IU/mg protein and gave a single protein band (apparent molecular weight; 67,000) in SDS-polyacrylamide gels in the presence or absence of a reducing agent. The increase in specific activity was 3,200-fold, most of which was achieved in the step of heparin-Sepharose chromatography. The yield calculated from the active ammonium sulfate precipitate was about 90% and 500 micrograms or more of the purified enzyme was obtained from 1 kg wet tissue. This procedure may also be useful for the large-scale production of highly purified t-PA from other tissues or tissue culture cells.
D000596:Amino Acids / Q000032:analysis; D000645:Ammonium Sulfate; D000818:Animals; D055598:Chemical Phenomena; D011232:Chemical Precipitation; D002621:Chemistry; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D008970:Molecular Weight; D009206:Myocardium / Q000032:analysis*; D011108:Polymers; D012685:Sepharose / Q000031:analogs & derivatives; D013552:Swine; D010959:Tissue Plasminogen Activator / Q000302:isolation & purification*
D000596:Amino Acids; D011108:Polymers; C037405:heparin-sepharose; C030724:Toyopearl HW55F; D012685:Sepharose; D010959:Tissue Plasminogen Activator; D000645:Ammonium Sulfate