Leslie Lekatz (Bemidji State) and William T. Doerrler, Biological Sciences

Cloning and overexpression of Imp/OstA, an essential Escherichia coli outer membrane protein with a possible role in lipid transport

Lipopolysaccharide (LPS), an essential component of the Escherichia coli (E. coli) outer membrane (OM) is synthesized inside the cell and must cross the inner membrane (IM), periplasm, and OM in order to reach their location in the cell, the outer surface of the OM. MsbA, an IM protein of the ABC transporter superfamily, is believed to play a role in the transport of newly synthesized LPS across the IM. The mechanism of transport of LPS across the outer membrane is not understood, but recent evidence suggests that Imp/OstA (increased membrane permeability/organic solvent tolerance), an essential and highly conserved E. coli OM protein, plays a role in lipid transport across the OM. This study was conducted to look more closely at Imp and its role in membrane biogenesis. The imp gene was amplified from E. coli genomic DNA by polymerase chain reaction and cloned into an expression vector behind a T7 promoter, which was then used to transform E. coli cells. Imp was overexpressed and localized correctly to the OM. The protein was resistant to solubilization by several detergents, even though other OM proteins were solubilized. This work begins our efforts to purify, crystallize and structurally characterize this novel OM protein.