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 process, 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 changed to glutamine and a third version with both mutations. These mutant streptokinase proteins (muteins) were produced by secretion with the protease-deficient Bacillus subtilis WB600 as the host. The purified muteins retained comparable kinetics parameters in plasminogen activation 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 with plasminogen in vitro and showed better plasminogen activation activity with time in the radial caseinolysis assay. This study indicates that plasmin-mediated processing leads to the inactivation of streptokinase and is not required to convert streptokinase to its active form. Plasmin-resistant forms of streptokinase can be engineered without affecting their activity, and blockage of the N-terminal cleavage site is essential to generate engineered streptokinase with a longer in vitro functional half-life.
Wu
X C
XC
Department of Biological Sciences, University of Calgary, Alberta, Canada.
Ye
R
R
Duan
Y
Y
Wong
S L
SL
D001412:Bacillus subtilis / Q000235:genetics*; D005341:Fibrinolysin / Q000494:pharmacology*; D006207:Half-Life; D016297:Mutagenesis, Site-Directed; D010958:Plasminogen / Q000378:metabolism; D015202:Protein Engineering; D013300:Streptokinase / Q000096:biosynthesis* / Q000235:genetics
D010958:Plasminogen; D013300:Streptokinase; D005341:Fibrinolysin