SECRETORY PRODUCTION OF RECOMBINANT UROKINASE-TYPE PLASMINOGEN ACTIVATOR-ANNEXIN V-CHIMERAS IN PICHIA-PASTORIS

To produce a thrombi-targeting plasminogen activator, we expressed a f used gene that contains a modified pre-sequence of Mucor pussilus renn in (MPR) followed by a chimeric gene of single-chain urokinase-type pl asminogen activator (scu-PA)::annexin V (AV). The fused gene was ligat ed into an integrative vector, under the control of the alcohol oxidas e 1 (AOX1) promoter (p), and transformed into Pichia pastoris. Transfo rmants were monitored for the secretion of fibrinolytic activity. The highest expressing clone, HB225, secreted as much as 600 international units (IU) of fibrinolytic activity per mi of culture medium under op timal conditions. It contained three tandem copies of the full-size ve ctor disruptively integrated into the AOX1 sequence. Western blot anal ysis revealed that the secreted chimera was highly susceptible to prot eolysis. Addition of excess amino acids (aa) to the culture medium min imized the degree of proteolysis. Two major species of chimera, 85 and 65 kDa, were then isolated from the culture medium. The former was th e intact form consisting of a single-chain and showing full enzyme act ivity after activation by plasmin. The latter was an enzymatically pro cessed form consisting of two chains held by a disulfide bond, having full enzyme activity without activation. Both chimeras exhibited calci um-dependent phospholipid (PL)-binding affinities similar to the paren t AV.