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Paper Information

Journal:   DANESHVAR MEDICINE   JUNE-JULY 2007 , Volume 14 , Number 69; Page(s) 61 To 68.
 
Paper: 

CLONING AND EXPRESSION OF STREPTOKINASE GENE WITH A 6XHIS LABEL IN E.COLI.

 
 
Author(s):  MEMARNEZHADIAN A., FOROUZANDEH MOGHADAM M., SADAT S.M., ROUHVAND F.*
 
* PASTEUR INSTITUTE OF IRAN, HEPATITIS AND AIDS UNIT
 
Abstract: 

Purpose: Streptokinase (SK), a naturally secreted plasminogen activator by groups A, C and G streptococci, is widely used for thrombolytic therapy. There is quite heterogeneity among the SK of different producer strains of the bacteria that may show different antigenicity and biological properties. Herein, with the aim of establishing an easy system for expression and purification of the recombinant SK (r-SK) from different strains, SK gene has been cloned.
Materials & Methods: SK gene from a standard strain (skc2) has been cloned as a PCR amplified fragment under control of T5 promoter into pQE30 vector, which adds a 6xHis-tag to the N-terminal of the protein, to simplify purification through application of Ni-NTA agarose column. Accuracy of the construct was confirmed by restriction mapping and E.coli M15 strain was exploited for expression of the SK gene.
Results and Discussion: Finally, production of the active r-SK reached to around 60% of total protein following induction by IPTG, under optimized condition, which was confirmed by SDSPAGE analysis and produced r-SK had a MW of 56 KD as determined empirically from Rf values of immigrated bands in the SDS-PAGE, which was identical to the expected theoretical values.
Conclusion: Results of this study suggested that this system might be successfully applied to express and purify different SK genes in high yield, without interfering their activity.

 
Keyword(s): STREPTOKINASE, HIS TAG, PQE30, PLASMINOGEN
 
References: 
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