Enhancement of streptokinase activity by site directed mutagenesis

Abstract

A bacterial plasminogen activator, streptokinase (SK) is widely used as a thrombolytic agent in the treatment of myocardicac infarction. In this study, streptokinase gene was modified by using the technique of directed evolution. Through six-rounds of random mutagenesis and three rounds of error-prone PCR, over 50 thousands of clones showing larger hollow than wild type SK were screened by skim-milk plasminogen overlay test. Among them, SK-S221F which showed the highest activity was purified and characterized. SK-S221F shows the lower Km value than wild type SK and showed 2.5 folds higher overall second order rate constant than wild type complex. It suggests that the SK-221F mutant could efficiently recruit substrate plasminogen. To improve the better activity of SK, the amino acid sequences in β4 strand were changed by site directed mutagenesis. Among them, SK-R219L was purified and characterized. It shows 3 folds higher activation activity. To confirm the mechanism, SK-R219L and SK-E219F were made and these mutants show 1.3 and 3.9 times higher activity than wild type SK. It suggests that these mutants efficiently recruited substrate plasminogen. The possible usefulness of SK-221F in developing thrombolytic agent was discussed