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

BACKGROUND: The multifunctional enzyme a-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. alpha-enolase on the surface of Staphylococcus aureus (S. 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. a-enolase, as an important surface antigen, is a potential vaccine or immunotherapeutic candidate. METHODS: For cloning and expression studies, a-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 degrees 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. RESULTS: The highest protein expression was observed when the a-enolase production was induced using the mixture of two different inducers at the same time. CONCLUSION: We cloned, expressed, and purified a-enolase from S. aureus which could be a significant step for applying the recombinant enzyme in biotechnological processes.