Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

Summary

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.

Abstract

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique applications for OMV where traditional nanoparticles are proving too difficult to synthesize. To date, all Gram-negative bacteria have been shown to produce OMV demonstrating packaging of a variety of cargo that includes small molecules, peptides, proteins and genetic material. Based on their diverse cargo, OMV are implicated in many biological processes ranging from cell-cell communication to gene transfer and delivery of virulence factors depending upon which bacteria are producing the OMV. Only recently have bacterial OMV become accessible for use across a wide range of applications through the development of techniques to control and direct packaging of recombinant proteins into OMV. This protocol describes a method for the production, purification, and use of enzyme packaged OMV providing for improved overall production of recombinant enzyme, increased vesiculation, and enhanced enzyme stability. Successful utilization of this protocol will result in the creation of a bacterial strain that simultaneously produces a recombinant protein and directs it for OMV encapsulation through creating a synthetic linkage between the recombinant protein and an outer membrane anchor protein. This protocol also details methods for isolating OMV from bacterial cultures as well as proper handling techniques and things to consider when adapting this protocol for use for other unique applications such as: pharmaceutical drug delivery, medical diagnostics, and environmental remediation.

Introduction

Presented here is a method for the design, production, and purification of enzyme-loaded bacterial outer membrane vesicles (OMV). OMV are small, primarily unilamellar, proteoliposomes that range in size from 30-200 nm^1,2. All Gram-negative and Gram-positive bacteria that have been studied to date have demonstrated release of either OMV or extracellular vesicles (EV) from their surface^3,4. The precise mechanism by which OMV are produced have yet to be fully elucidated due to the diverse bacterial populations that secrete them as well as the varying functions that they serve. OMV have been shown to transport a wide range of cargo from small molecul....

Protocol

1. Preparation of Plasmids

1. Prepare a plasmid (e.g., pET22) containing the anchor protein (OmpA) fused to a biorthogonal linkage domain (referred to in this protocol as anchor-ST), epitope tag (such as 6xHis, myc or FLAG tags for purification and identification), periplasmic localization tag, antibiotic resistance, and appropriate origin of replication based on the selected bacterial strain⁷.
   1. Extract plasmid DNA from overnight cultures using a commercially available DNA isolation kit following the manufacturer's protocol.
2. Prepare a plasmid (e.g., pACYC184) containing the enzyme/protein (PT....

Results

Simultaneous expression of two recombinant proteins, as is required for the OMV packaging strategy detailed in this protocol, can be accomplished through a number of different avenues. Here, a two vector system was utilized with compatible origins of replication and separate inducible gene cassettes. For the expression of the PTE-SC construct a commercial plasmid backbone (pACY184) was engineered to include an arabinose inducible gene cassette and a twin arginine periplasmic localization .......

Discussion

This protocol functions to demonstrate a representative directed packaging technique in which an enzyme of interest is produced and packaged into OMV by E. coli. As with many complex techniques there are multiple areas in which the protocol can be modified to accommodate for use in different unique applications, some of which are detailed below. While the mechanism of OMV packaging and enzyme encapsulation can be adapted to specific needs there are several steps within this protocol which are critical to its suc.......

Materials

Name	Company	Catalog Number	Comments
IPTG	Any		Always prepare fresh or aliquot and freeze.
L-arabinose	Any		Can be prepared ahead of time and stored at 4C.
Ampicillin	Any		Add immediately prior to use after media cools sufficiently from being autoclaved.
Chloramphenicol	Any		Add immediately prior to use after media cools sufficiently from being autoclaved.
TB/LB Culture Media	Any		Other growth medias will likely work similarly.
Triton X-100	Any		One of many potential suitable surfactants.
Baffled culture flasks	Any		The baffles promote higher levels of aeration.
CHES	Fisher Bioreagents	BP318-100	Optimal buffer used for paraoxon degredation (pH > 8).
Paraoxon	Chem Service	N-12816	Very toxic substance to be handled carefully and disposed of properly.
Syringe Filter 0.45 µm	Thermo Scientific	60183-221       (30 mm)	Filter diameter will depend on volume of sample.  Low protein binding membrane is critical.
Shaker incubator	New Brunswick	Excella E24	Precise temperature and mixing is essential for reproducable bacterial growth.
Sorvall Culture Centrifuge	Thermo Scientific	RC 5B PLUS	Large volume (500 mL) culture centrifuge capable of 7,000 x g.
Sorvall Ultracentrifuge	Thermo Scientific	WX Ultra 90	Capable of centrifugal forces ≥150,000 x g.
Ultracentrifuge Rotor	Thermo Scientific	AH-629	Ensure the proper rotor and tubes are used and that everything is properly balanced.
Ultra-Clear Ultracentrifuge Tubes        (25 x 89 mm)	Beckman Coulter	344058	Ensure no stress fractures are present prior to use and that tubes are presicely balanced.
Spectrophotometer	Tecan	Infinite M1000	Necessary for enzyme kinetic assays.
DLS / particle tracking	NanoSight	LM10	Necessary for OMV size distribution and concentration determination.
BL21(DE3)	NEB		Suitable bacterial expression strain.
pET22	EMD Millipore	69744-3	Other plasmids can be used in place of these.
pACYC184	NEB		Other plasmids can be used in place of these.
Gel Extraction Kit	Qiagen	28704	Example kit.