Exploring Outer Surface Protein A from <em>Borrelia afzelii</em>: Cloning, Expression, and Characterization for Zoonotic Disease Research

Lyme disease (LD), caused by Borrelia burgdorferi sensu lato, is transmitted through the bite of infected ticks. Outer surface protein A (OspA) is a major lipoprotein expressed by Borrelia species, playing a critical role in the colonization of the tick midgut. OspA is a key target in diagnostic and vaccine development due to its conserved nature across Borrelia species. This study focuses on the expression and characterization of soluble recombinant OspA protein from Borrelia afzelii in the Escherichia coli expression system and its subsequent purification and functionality evaluation. The OspA gene from B. afzelii strain BO23 was cloned into the pET-28a(+) expression vector and transformed into E. coli BL21 Star (DE3) cells. Protein expression was induced under various growth conditions, including differing induction times, media types (Luria broth and 2xYT), and glucose concentrations. Optimization efforts centered on enhancing the yield of soluble OspA. Protein purification was carried out using a HisTrap column, and the purified protein&#039;s reactivity was assessed via Western blot using commercial OspA monoclonal antibodies. The optimal conditions for OspA expression were determined to be five hours of induction with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) in LB broth supplemented with 0.2% glucose, at 37°C. This has resulted in high yields of soluble OspA (12 mg/mL). Purified OspA demonstrated strong immuno reactivity to anti-OspA monoclonal antibodies, confirming its ability to bind to active epitope of OspA protein. This study successfully optimized the expression and purification of functional OspA in E. coli, providing a valuable resource for further Lyme disease research, including diagnostics and vaccine development.