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 in vasiveness and disease. SK activates Pg to plasmin (Ps) by forming an activator complex with Pg. However, the molecular basis whereby SK bin ds and activates Pg remains unclear, in part due to the rapid fragment ation of the SK-Pg complex. This study describes a solid phase approac h to study this interaction in which a recombinant SK molecule was con structed with glutathione-S-transferase appended to the NH2 terminus a nd (Gly)(3)(His)(8) appended to the COOH terminus. This dual fusion pr otein molecule, immobilized on either Sepharose-S-hexylglutathione or Ni2+ dinitriloacetic acid-Sepharose was then used to study the interac tion of SK with Pg. These SK-Pg complexes exhibited amidolytic and pro teolytic activity similar to native SK, but the pattern of fragmentati on of the SK molecule was dependent upon whether the SK molecule was i mmobilized either at its NH2- or COOH terminus. This solid phase appro ach may contribute to a greater understanding of the role of SK in Pg activation by enabling the 'capture' of intact activator complexes und er physiological conditions and, in addition, may serve as a useful mo del to analyze other bacterial-host protein interactions important in the pathogenesis of disease.