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A Journal on Biotechnology and Molecular Biology
Indexed/Abstracted in: EMBASE, Science Citation Index Expanded (SciSearch), Scopus
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Minerva Biotecnologica 2017 March;29(1):8-13
Copyright © 2014 EDIZIONI MINERVA MEDICA
Cloning, purification and enzymatic assay of streptokinase gene from Streptococcus pyogenes in Escherichia coli
Mohammad J. RAEE 1, 2, Abdollah GHASEMIAN 3, Samira MAGHAMI 1, 2, Mohammad B. GHOSHOON 1, 2, Younes GHASEMI 1, 2 ✉
1 Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran; 2 Department of Pharmaceutical Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran; 3 Department of Pharmaceutical Biotechnology, School of Pharmacy, Urmia University of Medical Sciences, Urmia, Iran
BACKGROUND: Streptokinase is an important therapeutic enzyme that acts as a fibrinolytic agent and use in the treatment of multi serious and life threatening diseases such as pulmonary embolism and acute myocardial infarction.
METHODS: Cloning and extracellular expression of recombinant streptokinase from native Streptococcus pyogenes PTCC 1447 was done by fusing the gene coding for streptokinase to an efficient vector (pET15b) with NdeI and BamHI as restriction enzymes and T4 DNA ligase. Streptokinase activity was determined using synthetic chromogenic substrate S-2251. Purification was done under N-terminal 6× histidine tag complementation using affinity chromatography.
RESULTS: The DNA and amino acid sequence alignments resulting from the BLAST search of streptokinase showed high sequence identity with the other strains of S. pyogenes. The recombinant enzyme was purified successfully showing high streptokinase activity.
CONCLUSIONS: Despite several limitations, streptokinase has some advantages such as low cost, available microbial source, and higher half-life in comparison with many other fibrinolytic agents.
KEY WORDS: Escherichia coli - Fibrinolytic agents - Molecular cloning - Streptococcus pyogenes - Streptokinase