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 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.

FIG. 1

Production and purification of streptokinase and its muteins. (a) Western blot of secreted streptokinase. Lanes: 1, prestained markers, with the molecular mass in kilodaltons shown on the left; 2 to 6, 60 μl of culture supernatant from WB600(pUB), WB600(pSK3), WB600(pSKC32), WB600(pSKN460), and WB600(pSKN460-C32), respectively. (b) SDS-PAGE analysis of purified streptokinase. Lanes: 1, molecular mass markers; 2 to 5, 5 μl of purified SKN460, SKC32, SKN460-C32 and natural streptokinase, respectively.

FIG. 2

Processing of streptokinase and its muteins by plasmin. Streptokinase and plasminogen were mixed in a 1:1 molar ratio and incubated at 37°C. Samples were collected at different time points (in minutes) and analyzed by Western blot with streptokinase-specific polyclonal antibodies. (a) Wild-type streptokinase; (b) SKN460; (c) SKN460-C32. An asterisk marks a new form of intermediate generated during the processing of SKN460.

FIG. 3

Activity of various forms streptokinase based on the radial caseinolysis. Each form of streptokinase, in equal quantities, was loaded into individual wells and incubated at 37°C for 12 h. Numbers indicate the relative activity of each form of streptokinase.