| Summary: | 碩士 === 慈濟大學 === 藥理暨毒理學研究所 === 98 === Streptokinase (SK), a currently used antithrombotic agent, was regarded as a fibrin-nonspecific plasminogen activator compared with tissue-type plasminogen activator (t-PA). A recent study found that removal of the N-terminal residues 1-59 allowed the remaining molecule (SK△59) to cause plasminogen activation in a fibrin-dependent manner, suggesting that SK△59 harbors the potenetial to become a novel thrombolytic agent. This study aimed to mass-produce SK△59, which allowed us to study further the action mechanism of SK△59 in lysing thrombi.
The DNA segment corresponding to the SK△59 was amplified by PCR and cloned into the expression vector pGEX-2T, which allowed SK△59 to be produced in fusion to the C-terminus of glutathione S-transferase (GST) in Escherischia coli. The successful transformant was grown in LB culture medium with additive of ampicillin (100 μg/ml) and glucose (20 mM). As the cells reached mid-log growth, IPTG (0.2 mM) was added to trigger GST-SK△59 expression for 5 hours. After that, cells were harvested, disrupted by sonication, and the GST-SK△59 fusion protein in the soluble fraction was purified with glutathione Sepharose 4B affinity column followed by anion exchange column chromatography. The GST part was then removed by thrombin cleavage.
Purified SK△59 exhibited a high purity when analyzed by SDS-PAGE. Chromogenic substrate assay revealed that SK△59 was less potent than SK in converting glu-plasminogen into plasmin, SK△59 was not as good as SK in triggering fibrin clot lysis either. These results are not consistant with previous observations by other investigators.
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