Engineering of Plasmin-Resistant Forms of Streptokinase and Their Production in Bacillus subtilis : Streptokinase with Longer Functional Half-Life

Abstract

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 subtilisWB600 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.

Bioactive protein fraction DLBS1033 containing lumbrokinase isolated from Lumbricus rubellus: ex vivo, in vivo, and pharmaceutic studies

“…The short in vivo half-life of currently available thrombolytic agents, such as streptokinase (30 minutes) and tissue plasminogen activator (tPA) (5 minutes) often limit their efficacies as an efficient blood clot dissolving agent. 20 Thus, with longer biological half-life (70 minutes), DLBS1033 could be more effective for thrombolytic therapy.…”

Development and Testing of Thrombolytics in Stroke

“…The very short half-life of streptokinase can be extended up to 21-fold by substituting specific charged residues responsible for its proteolysis or by PEGylation (the process of attaching polyethylene glycol [PEG] polymer to molecules). [108][109][110] Staphylokinase can be induced to have reduced immunogenicity (30%) or improved fibrinolytic effectivity by substituting only a few specific residues and using PEGylation. 111,112 Alteplase can be made resistant to PAI-1 by mutating four charged residues in the catalytic domain to alanine.…”

Structural Biology and Protein Engineering of Thrombolytics

“…Strategies were based on finding that during the process of plasminogen activation, the generated plasmin non-physiologically cleaves streptokinase into three polypeptide chains (residues 1–59, 60–386, and 387–414) leading to drop in activity [233,234]. Preventing the cleavage by mutagenesis at the identified positions (Lys59Gln/Glu, Lys386Gln) resulted in a 21-fold increased half-life without affecting activity [[235], [236], [237]]. Alternative strategies based on combinatorial mutagenesis [14,238], PEGylation [[239], [240], [241], [242]], glycosylation [235,243], or lipidification [244] yielded streptokinase variants with improved thrombolytic activity, decreased immunogenicity, and higher half-life.…”