Expression, purification and characterization of hemin-binding lipoprotein PiuA from Streptococcus pneumoniae

Abstract:

[Background] Streptococcus pneumoniae is one of the most common pathogens of community-acquired pneumonia, and causes a variety of other serious diseases including bacterial meningitis, sinusitis, otitis media and bacteremia, and poses health threats to humans, especially among children and the elderly, or individuals with immature/compromised immune systems. Iron is essential for Streptococcal survival and infection, and haem ABC uptake system PiuABCD is the most iron ABC uptake system for S. pneumoniae. [Objective] Clone, express and purify hemin-binding lipoprotein PiuA belonging to haem ABC uptake system PiuABCD, and characterize its hemin-binding properties in vitro. [Methods] The piuA (spd_1652) gene from S. pneumoniae D39 strain was ligated into pBAD-HisA vector, and expressed in E. coli Top 10 strain, then the PiuA-His fusion protein was purified using Ni-NTA affinity chromatography, and the His tag free PiuA protein was obtained by cutting off the His tag with enterokinase. Finally, the hemin-binding properties of PiuA protein were characterized by circular dichroism, UV spectroscopy and fluorescence spectroscopy. [Results] We successfully constructed recombinant vector pBAD/HisA-piuA, and acquired the PiuA protein with the purity higher than 95%. CD spectra showed that hemin binding did not induce any significant structural change in PiuA, UV spectroscopy revealed that PiuA protein can specifically bind hemin, and fluorescence spectroscopy demonstrated that the binding constant K between the PiuA and hemin is 3.4×105 L/mol. [Conclusion] Hemin-binding lipoprotein PiuA can specifically bind to hemin and provide the iron for Streptococcal survival and infection, these characterization results of PiuA protein provide basis data for design of antibacterial drug targeted for haem ABC uptake system PiuABCD.