Cloning, purification and enzymatic assay of streptokinase gene from Streptococcus pyogenes in Escherichia coli

Streptokinase is an important therapeutic enzyme that acts as a fibrinolytic agent and
use in the treatment of multi serious and life threatening diseases such as pulmonary embolism and acute
myocardial infarction. Cloning and extracellular expression of recombinant streptokinase from Streptococcus
pyogenes was done by fusing the gene coding for streptokinase to an efficient vector (pET15b) with NdeI and
BamHI as restriction enzymes and T4 DNA ligase. Streptokinase activity was determined using synthetic
chromogenic substrate S-2251, and measuring the absorbance at 405 nm in a UV-spectrophotometer; and
purification done under N-terminal 6× histidine tag complementation using affinity chromatography.
A 1323-bp of the structural streptokinase gene was amplified. The DNA and amino acid sequence alignments
resulting from the BLAST search of streptokinase showed high sequence identity with the other strains of S.
pyogenes. The recombinant enzyme had the same molecular weight as other reported streptokinase and the E.
coli transformants showed high streptokinase activity.
Comparative clinical trials and cost-effectiveness considerations between several major available thrombolytic
agents suggest that streptokinase is the drug of choice for thrombolytic therapy particularly in the poorer
economies.