Molecular Design, Cloning, Expression and Purification of NH(2)-terminal Mutant of Staphylokinase deltaNSak
The 3-D structure of human microplasminogen was predicted by the method of homology modeling. The binding sites of staphylokinase and microplasminogen were determined by high-resolution genetic docking. This model is consistent with several known experimental properties of staphylokinase. To elucidate the function of NH(2)-terminus of staphylokinase for further rational reconstruction, a NH(2)-terminal-deletion mutant of staphylokinase(deltaNSak) was designed. The soluble deltaNSak was achieved with fibrinolytic activity. deltaNSak was purified by two-step ion exchange chromatography. The purity was over 95% and the specific activity of deltaNSak was 9x10(4) IU/mg. These data suggest that the 15 NH(2)-terminal amino acids of staphylokinase are not required for its plasminogen activating potential and the computer model is reasonable.
Department of Molecular Genetics, School of Basic Medical Sciences, Shanghai Medical University, Shanghai 200032, China. hysong@shmu.edu.cn
Gang
G
Song
Wei
W
Mo
Hou-Yan
HY
Song