Using biotinylation to determine the orientations of Pal in Escherichia coli

Abstract

Nontypable Haemophilus influenzae (NTHi) causes otitis media and other respiratory diseases in people of all ages, conferring the need for a vaccine against the bacteria. P6, a lipoprotein vaccine candidate for NTHi, was recently discovered to exhibit two populations: one facing out of the cell and one facing in towards the periplasm. We hypothesized that dual orientation is a novel, widespread phenomenon among bacterial lipoproteins. To corroborate our hypothesis, we performed a biotinylation experiment on Pal, homolog to P6, in Escherichia coli (E. coli). In the experiment, whole cells were incubated with a cell‐impermeable biotinylating reagent (NHS‐LC‐LC‐biotin) in order to label all surface exposed proteins. The labeled, outer proteins were separated from the unlabeled, internal proteins via streptavidin‐agarose beads. Standard protein detection and immunoblotting techniques confirmed that Pal exists in two orientations in E. coli, with the majority of Pal facing in toward the periplasmic space. Only one other protein (Lpp of E. coli) has been shown to exhibit dual orientations. The results of this experiment suggest that lipoprotein dual orientation may be more widespread than originally thought, which could have important implications in the field of bacterial lipoprotein biology. This study was funded by the Rochester Institute of Technology and NIH NIDCD RO1 08671 (to MEP).