STREPTOKINASE-MEDIATED PLASMINOGEN ACTIVATION USING A RECOMBINANT DUAL FUSION PROTEIN CONSTRUCT - A NOVEL-APPROACH TO STUDY BACTERIAL HOST PROTEIN INTERACTIONS

Streptokinase (SK), a plasminogen (Pg) activator secreted by groups A, C, and G streptococci, is extensively used as a pharmacological agent in thrombolytic therapy and possibly plays a role in streptococcal invasiveness and disease. SK activates Pg to plasmin (Ps) by forming an activator complex with Pg. However, the molecular basis whereby SK binds and activates Pg remains unclear, in part due to the rapid fragmentation of the SK-Pg complex. This study describes a solid phase approach to study this interaction in which a recombinant SK molecule was constructed with glutathione-S-transferase appended to the NH2 terminus and (Gly)(3)(His)(8) appended to the COOH terminus. This dual fusion protein molecule, immobilized on either Sepharose-S-hexylglutathione or Ni2+ dinitriloacetic acid-Sepharose was then used to study the interaction of SK with Pg. These SK-Pg complexes exhibited amidolytic and proteolytic activity similar to native SK, but the pattern of fragmentation of the SK molecule was dependent upon whether the SK molecule was immobilized either at its NH2- or COOH terminus. This solid phase approach may contribute to a greater understanding of the role of SK in Pg activation by enabling the 'capture' of intact activator complexes under physiological conditions and, in addition, may serve as a useful model to analyze other bacterial-host protein interactions important in the pathogenesis of disease.