Cloning and expression of truncated and intact streptokinase molecules in E.coli and evaluation of their biological activities
Streptokinase is the most utilized drug for thrombolytic therapy. However, due to its nonhuman origin and nonspecific activity in the absence of fibrin, its application may encounter side effects such as allergic reactions and hemorrhage respectively. It is suggested that some truncated forms of str...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | fas |
| Published: |
Shahid Bahonar University of Kerman
2010-05-01
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| Series: | مجله بیوتکنولوژی کشاورزی |
| Subjects: | |
| Online Access: | https://jab.uk.ac.ir/article_361_3a5691e6e2d2a571fde84d88c5d12964.pdf |
| Summary: | Streptokinase is the most utilized drug for thrombolytic therapy. However, due to its nonhuman origin and nonspecific activity in the absence of fibrin, its application may encounter side effects such as allergic reactions and hemorrhage respectively. It is suggested that some truncated forms of streptokinase may show better fibrin-specificity and decreased antigenicity while retaining most of the enzymatic activity. In the present study, different truncated forms of streptokinase gene,”<em>Skc</em>60-386 and <em>skc</em>143-386”together with the intact form of <em>skc</em> (<em>skc</em>1-414)were cloned in <em>T5</em> and <em>T7</em> based-<em>E.coli</em> expression vectors, “pQE30 and pET41a” respectively. While expression of intact streptokinase could equally be achieved in both systems, but pET41a was the only effective system to produce truncated forms. pET41a vector contains three tandem tags before multiple cloning sites which possibly prevent intrinsic problems encountered in heterologous recombinant protein expression in <em>E.coli</em>. Analyses of the purified proteins by SDS-PAGE and Western blotting evidenced for the true expression and the expected MWs. While caseinolysis method was not able to demonstrate exact difference between specific activity of SK143-386, SK60-386 and SK1-414but truncated molecules showed high reduction of specific activity (83 to 91 percent) compared to the full-length streptokinase by more exact techniques such as colorimetric assay. This study provided a primary comparative study for expression of streptokinase and its truncated forms and analyses of their specific activity. Thus further analyses would determine their specificity to fibrin and antigenicity. |
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| ISSN: | 2228-6705 2228-6500 |