Large-scale purification of human tissue-type plasminogen activator using monoclonal antibodies.
Tissue plasminogen activator produced by a human melanoma cell line (Bowes), was purified from large volumes of supernatant fluid using immunosorbent chromatography on monoclonal antibodies, followed by chromatography on lysine-Sepharose 4B and gel filtration on Sephadex G-150. Immunosorbents based on monoclonal antibodies were shown to be preferable to those based on polyclonal antibodies for several reasons: efficient binding and desorption, high yield along with a high degree of purification. The overall recovery was about 50%. A homogeneous sample of single-chain tissue plasminogen activator with a molecular weight of approx. 65 000 was obtained as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Amino acid sequence analysis revealed two N-terminals in equal yields, corresponding to the long and short tissue plasminogen activator structures previously reported by others. The pure preparations of tissue plasminogen activator showed specific activities of about 200 000 IU/mg protein.
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D002460:Cell Line; D002845:Chromatography; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008545:Melanoma; D008970:Molecular Weight; D010960:Plasminogen Activators / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D010960:Plasminogen Activators