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Large‐Scale Purification of Tissue‐Type Plasminogen Activator from Cultured Human Cells
Author(s) -
Harakas Nikos K.,
Schaumann Jon P.,
Connolly Daniel T.,
Wittwer Arthur J.,
Olander Jitka V.,
Feder Joseph
Publication year - 1988
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1002/btpr.5420040305
Subject(s) - affinity chromatography , sepharose , concanavalin a , chemistry , plasminogen activator , chromatography , monoclonal antibody , agarose , size exclusion chromatography , enzyme , specific activity , biochemistry , antibody , biology , in vitro , immunology , endocrinology
Plasminogen activators of both urokinase‐type (u‐PA) and tissue‐type (t‐PA) have been found in conditioned medium of cultured human fibroblasts. To purify t‐PA, the total PA activity from hectoliter quantities of conditioned medium was first concentrated with about 10‐fold increase in specific activity via zinc chelate‐sepharose affinity chromatography. Three different methods were employed in an attempt to separate t‐PA from u‐PA: (a) concanavalin A‐sepharose affinity chromatography; (b) fibrin‐celite affinity chromatography; and (c) immunoaffinity chromatography using anti‐tPA monoclonal antibody immobilized on sepharose. Of the three methods, only the monoclonal antibody procedure effected complete separation of the two enzymes. t‐PA was subsequently purified to homogeneity using size exclusion, high performance liquid chromatography followed by p‐aminobenzamidine‐agarose affinity chromatography.