Enhancement of streptokinase activity by directed evolution

Abstract

A bacterial plasminogen activator, streptokinase is widely used as a thrombolytic agent in the treatment of myocardial infarction. In this study, streptokinase was modified by directed evolution. Through six rounds of random mutagenesis and three rounds of error-prone PCR, over 50 thousands of clones were screened by skim milk-plasminogen overlay test and plasminogen activation assay in 96-well plates. As a result, seven mutants with amino acid substitution were selected. Among them, S221F was purified and its amidolytic activity and plasminogen activation activity were analyzed at pH 7.4 and 37℃ with a chromogenic substrate, $NH_{2-D}-Val-Leu-Lys-p-nitroanilide. The S221F showed the 7.7-fold increased specific activity than wild-type SK owing to the decreased Michaelis constant for the substrate plasminogen. The evolved F221 may contribute to a hydrophobic core of SK β domain for substrate plasminogen binding.