ENGINEERING OF PLASMIN-RESISTANT FORMS OF STREPTOKINASE AND THEIR PRODUCTION IN BACILLUS-SUBTILIS - STREPTOKINASE WITH LONGER FUNCTIONAL HALF-LIFE

The short in vivo half-life of streptokinase limits its efficacy as an efficient blood clot-dissolving agent, During the clot-dissolving pro cess, streptokinase is processed to smaller intermediates by plasmin, Two of the major processing sites are Lys59 and Lys386, We engineered two versions of streptokinase with either one of the lysine residues c hanged to glutamine and a third version with both mutations. These mut ant streptokinase proteins (muteins) were produced by secretion with t he protease-deficient Bacillus subtilis WB600 as the host. The purifie d muteins retained comparable kinetics parameters in plasminogen activ ation and showed different degrees of resistance to plasmin depending on the nature of the mutation, Muteins with double mutations had half- lives that were extended 21-fold when assayed in a 1:1 molar ratio wit h plasminogen in vitro and showed better plasminogen activation activi ty with time in the radial caseinolysis assay. This study indicates th at plasmin-mediated processing leads to the inactivation of streptokin ase and is not required to convert streptokinase to its active form. P lasmin-resistant forms of streptokinase can be engineered without affe cting their activity, and blockage of the N-terminal cleavage site is essential to generate engineered streptokinase with a longer in vitro functional half-life.