کلون، بیان، خالص سازی و بهینه سازی بیان آنزیم آلفا-انولاز از باکتری استافیلوکوکوس اورئوس در ایشرشیاکولای

عنوان انگليسی

Cloning, expression, purification and expression condition optimization of α-enolase from Staphylococcus aureus in Escherichia coli

خلاصه انگلیسی

The multifunctional enzyme α-enolase belongs to a family of cytoplasmic and glycolytic enzymes, which is localized in the cytoplasm and at the surface of many eukaryotic and prokaryotic cells. α-enolase on the surface of Staphylococcus aureus is a receptor with high affinity for laminin, the most abundant extracellular matrix component. Laminin-binding ability plays a considerable role in the pathogenesis of this microorganism. S. aureus is an opportunistic pathogen that causes major nosocomial infections and variety of diseases in human beings. This organism has a strong tendency to develop antibiotic resistance. Its property to spread of antibiotic resistance has intensified the need of novel antistaphylococcal techniques. α-enolase as an important surface antigen, is a potential vaccine or immunotherapeutic candidate. For cloning and expression studies, α-enolase gene was amplified by PCR using the specific primers. Then it was inserted into the pET-15b vector and transformed in to Escherichia coli DE3. Gene expression was induced at 30°C using IPTG and the recombinant enzyme purification carried out by Ni-NTA Spin Columns. Protein was shown with SDS-PAGE analysis and then, different induction conditions for optimization of the recombinant protein expression were used. The highest protein expression was observed when the α-enolase production was induced using the mixture of two different inducers at the same time.

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