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

Koki Kamiya

doi:10.1038/s41598-022-06314-4

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.

Highlights

Outer membrane proteins (OMPs) are inserted into the outer membrane of gram-negative bacteria[1,2]
A band of OmpG was observed at approximately 30 kDa, which was not observed in the solution of the in vitro translation system without OmpG DNA (Fig. 2a, Figure S1)
We show that OmpG incorporated into nano-sized liposomes can be quantified by stepwise sucrose density gradient, which can remove the liposome-free OmpG and other molecules of the in vitro translation system from the OmpG-containing liposomes

Summary

Introduction

Outer membrane proteins (OMPs) are inserted into the outer membrane of gram-negative bacteria[1,2]. Functional membrane proteins can be purified using the in vitro translation solution, that includes the nano-sized liposomes, as the membrane proteins are inserted directly into the liposomes during membrane protein s ynthesis[14]. The lipid membrane insertion of OmpA and OmpG using the in vitro translation system is investigated based on the composition of the liposomes and the presence of periplasmic chaperones such as Skp, DegP, and SurA11,12. The incorporation of OmpG into nano-sized liposomes, containing various lipid compositions, is investigated using purified nano-sized liposomes after in vitro translation (Fig. 1). To investigate the nanopore formation of OmpG synthesized by in vitro translation, the ion currents of OmpG, which is incorporated into a BLM by fusing between the OmpG liposome and the BLM, are measured in the artificial cell membranes using the patch clamp system

Methods

Results

Conclusion