[Expression, purification and characterization of a novel staphylokinase with anti-platelet aggregation activity].
OBJECTIVE
To investigate construction and expression of novel staphylokinase (SAK) mutants with Arg-Gly-Asp (RGD) motif at its N-terminus, and to develop a novel thrombolytic agent with fibrinolytic activity and anti-platelet activity.


METHODS
The fragments of SAK mutants were inserted into expression plasmid pBV220 and recombinant vectors were transformed into E. coli BL21, The SAK mutants could be expressed by rose of the culture temperature from 30 degrees C to 42 degrees C. After purified by a 3-step chromatography, their fibrinolytic and anti-platelet aggregation activity were analyzed.


RESULTS
SDS-PAGE analysis indicated that the SAK mutants occupied 50% of the total proteins in E. coli BL21. After purified by the 3-step chromatography, the purity of SAK mutants were more than 95%. The Specific fibrinolytic activities of SAK mutants purified were 10.8 x 10(4) and 11.0 x 10(4) HU x mg(-1). Anti-platelet aggregation activity were 10.72% and 19.71%, which were significantly higher than that of wild-type SAK. Thus the SAK variants were successfully expressed, and purified. The high purity and bioactivities of staphylokinase variants lay a good basis for manufacture and clinical application.
Jingfang
Di
D
Department of microbiology, College of Life Sciences, Hebei Normal University, Shijiazhuang 050016, China. fangfangxiaodi@yahoo.com.cn
Jintian
He
H
Ruiguang
Xu
X
Xi
Chen
C
Baohua
Zhao
Z
D020816:Amino Acid Motifs; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D009154:Mutation; D010974:Platelet Aggregation; D010975:Platelet Aggregation Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D010975:Platelet Aggregation Inhibitors; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; C022653:auR protein, Staphylococcus aureus