Formation and function of OmpG or OmpA-incorporated liposomes using an in vitro translation system

Abstract

Outer membrane proteins (OMPs), located on the outer membrane of gram-negative bacteria, have a β-strand structure and form nanopores, which allow passage of ions, sugars, and small molecules. Recently, OMPs have been used as sensing elements to detect biological molecules. OMPs are normally expressed and purified from Escherichia coli (E. coli). Although the cell-free synthesis of OMPs, such as OmpA and OmpG, is achieved in the presence of liposomes and periplasmic chaperones, the amount of OmpA and OmpG incorporated into the nano-sized liposomes is not clear. In this study, after in vitro translation, the incorporation of OmpG into purified nano-sized liposomes with various lipid compositions was investigated. Liposomes containing the synthesized OmpG were purified using a stepwise sucrose density gradient. We report that liposomes prepared with the E. coli lipid extract (PE/PG) had the highest amount of OmpG incorporated compared to liposomes with other lipid compositions, as detected by recording the current across the OmpG containing liposomes using the patch clamp technique. This study reveals some of the requirements for the insertion and refolding of OMPs synthesized by the in vitro translation system into lipid membranes, which plays a role in the biological sensing of various molecules.

Figure 1

Schematic representation of formation and function of OmpG liposomes using the in vitro translation system.

Figure 2

(a) SDS-PAGE analysis and (b) western blot analysis of OmpG synthesized by the in vitro translation system. Lane 1: protein molecular weight ladder, lane 2: Purefrex solution (+ OmpG encoded DNA) with nano-sized liposomes, lane 3: Purefrex solution (+ OmpG encoded DNA), lane 4: Purefrex solution (− OmpG encoded DNA).

Figure 3

SDS-PAGE analysis of before (a) and after (b) stepwise sucrose density gradient purification of OmpG synthesis containing nano-sized liposomes. Lane 1: protein molecular weight ladder, lane 2: Purefrex solution (+ OmpG encoded DNA) with DOPC liposomes, lane 3: Purefrex solution (+ OmpG encoded DNA) with DLPC liposomes, lane 4: Purefrex solution (+ OmpG encoded DNA) with Escherichia coli (E. coli) lipid liposomes, lane 5: Purefrex solution (+ OmpG encoded DNA) with DOPE/DOPG liposomes, and (a) lane 6: Purefrex solution (+ OmpG encoded DNA) or (b) lane 6: purified OmpG expressed by E. coli. (c) Protein amount into nano-sized liposomes. (d) SDS-PAGE analysis of OmpG into nano-sized liposomes by addition of nano-sized liposomes before or after OmpG synthesis. Lane 1: protein molecular weight ladder, lane 2: Purefrex solution (+ OmpG encoded DNA) by adding E. coli lipid liposomes before OmpG synthesis, lane 3: Purefrex solution (+ OmpG encoded DNA) by adding DOPE/DOPG liposomes before OmpG synthesis, lane 4: Purefrex solution (+ OmpG encoded DNA) by adding E. coli lipid liposomes after OmpG synthesis, lane 5: Purefrex solution (+ OmpG encoded DNA) by adding DOPE/DOPG liposomes after OmpG synthesis, and lane 6: purified OmpG expressed by E. coli. Black triangles represent OmpG band. Although there is separation between lane1–4 and lane5,6, the same gel of lane1–6 was conducted.

Figure 4

(a) Single channel current recording of OmpG incorporated into nano-sized liposomes (applied voltage: + 100 mV). (b) Histogram of OmpG current amplitude.