Abstract
Shigella spp. invade the colonic epithelium and cause bacillary dysentery in humans. Individuals living in areas that lack access to clean water and sanitation are the most affected. Even though infection can be treated with antibiotics, Shigella antimicrobial drug resistance complicates clinical management. Despite decades of effort, there are no licensed vaccines to prevent shigellosis. The highly conserved invasion plasmid antigens (Ipa), which are components of the Shigella type III secretion system, participate in bacterial epithelial cell invasion and have been pursued as vaccine targets. However, expression and purification of these proteins in conventional cell-based systems have been challenging due to solubility issues and extremely low recovery yields. These difficulties have impeded manufacturing and clinical advancement. In this study, we describe a new method to express Ipa proteins using the Xpress+TM cell-free protein synthesis (CFPS) platform. Both IpaB and the C-terminal domain of IpaH1.4 (IpaH-CTD) were efficiently produced with this technology at yields > 200 mg/L. Furthermore, the expression was linearly scaled in a bioreactor under controlled conditions, and proteins were successfully purified using multimode column chromatography to > 95% purity as determined by SDS-PAGE. Biophysical characterization of the cell-free synthetized IpaB and IpaH-CTD using SEC-MALS analysis showed well-defined oligomeric states of the proteins in solution. Functional analysis revealed similar immunoreactivity as compared to antigens purified from E. coli. These results demonstrate the efficiency of CFPS for Shigella protein production; the practicality and scalability of this method will facilitate production of antigens for Shigella vaccine development and immunological analysis.Key points• First report of Shigella IpaB and IpaH produced at high purity and yield using CFPS• CFPS-IpaB and IpaH perform similarly to E. coli–produced proteins in immunoassays• CFPS-IpaB and IpaH react with Shigella-specific human antibodies and are immunogenic in mice.Graphical abstract
Highlights
Shigella spp. are a leading cause of moderate to severe diarrhea in young children living in low- and middle-income countries and the second cause of diarrheal mortality among all ages (Khalil et al 2018; Kotloff et al 2019; Livio et al 2014)
The co-eluting chaperone was effectively removed from IpaB, by washing the column bound protein fraction with loading buffer supplemented with the zwitterionic detergent lauryl diamine oxide (LDAO)
SEC-Multi‐angle light scattering (MALS) analysis performed on purified IpaB showed that the cell-free protein synthesis (CFPS)-produced IpaB had a molecular mass of 121.5 ± 0.2 kDa, which is a close approximation to the theoretical mass of 124.2 kDa for an IpaB dimer
Summary
Shigella spp. are a leading cause of moderate to severe diarrhea in young children living in low- and middle-income countries and the second cause of diarrheal mortality among all ages (Khalil et al 2018; Kotloff et al 2019; Livio et al 2014). A hindrance to this endeavor has been the incomplete understanding of bacterial pathogenesis, the underlying mechanisms of host defenses, and antigen specificity required for protective immunity. Bacterial polysaccharides require bystander T helper–inducing molecules to generate strong and long-lasting adaptive immunity (Avci et al 2019; Rappuoli 2018). These requirements increase complexity of manufacturing and cost. The Ipa proteins are part of the Shigella type III secretion system (T3SS), a molecular machine that injects bacterial virulence effectors into host cells (Fig. 1) (Bajunaid et al 2020; Schnupf and Sansonetti 2019; Schroeder and Hilbi 2008), a critical initial step in Shigella invasion of colonic epithelial cells
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
