Large scale, rapid purification of recombinant tissue-type plasminogen activator.
Recombinant tissue-type plasminogen activator (rt-PA) from cultures of a genetically manipulated Bowes melanoma cell line (TRBM6) was purified in batches of average volume 451 using an autoclavable, reusable, continuous chromatography system comprising zinc chelate-Sepharose CL4B and lysine-Sepharose CL4B. After eight successive purifications the rt-PA was ultrafiltered to yield a preparation containing 4.9 mg protein/ml and 2.7 X 10(6) IU/ml. Analysis by SDS-polyacrylamide gel electrophoresis followed by staining with Coomassie brilliant blue R250 showed major protein bands at Mr = 63,000 and 65,000; most of the material was in the 1-chain form. The potential usefulness of a simple, rapid continuous chromatography system that can be operated under aseptic conditions is discussed.
D000818:Animals; D002460:Cell Line; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D008546:Melanoma, Experimental; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000302:isolation & purification*; D010959:Tissue Plasminogen Activator / Q000302:isolation & purification*
D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator