Production of Virus-like Particles in Engineered E. coli

Abstract

Source: Chen, Z., et al. Making Conjugation-induced Fluorescent PEGylated Virus-like Particles by Dibromomaleimide-disulfide Chemistry. J. Vis. Exp. (2018)

This video demonstrates the method for the controlled expression of the bacteriophage Qβ coat protein in engineered E. coli. The resulting proteins self-assemble into virus-like particles, which are harvested for downstream bioconjugation.

Protocol

Expression of Qβ

1. Wipe bench area with 1:1 bleach/ethanol.
2. Make two 3 mL starter cultures in an aseptic environment by adding single colonies of E. coli BL21(DE3) into 3 mL of Super Optimal Broth (SOB) media in an aseptic environment.
3. Grow on a shaker at 250 rpm in a 37 °C and 0% relative humidity (rH) room overnight.
4. Inoculate starter culture in SOB media:
   1. Take both 3 mL starter cultures off the shaker and, in an aseptic environment, pour each starter culture into one of two 2 L baffled Erlenmeyer flasks with 1 L of fresh SOB media in each.
   2. Place the inoculated media on a shaker at 250 rpm in a 37 °C and 0% rH room.
5. Grow the bacteria on a shaker at 250 rpm in a 37 °C and 0% rH room until OD₆₀₀ reaches 0.9–1.0.
6. Add 1 mL of 1 M isopropyl β-D-1 thiogalactopyranoside (IPTG) using a P1000 pipette to induce protein expression.
7. Leave the media on the shaker at 250 rpm in a 37 °C and 0% rH room overnight.
8. Remove media from the shaker the following morning and centrifuge it using 1000 mL bottles at 20,621 × g for 1 h at 4 °C to harvest the cells.
9. Discard the supernatant and collect the cell pellet.
   1. Pour the supernatant into a flask with about 5 mL of bleach to kill bacteria. This is a waste.
   2. Use a spatula to scrape the cell pellet from the bottom of the centrifuge bottle and put the pellet into a 50 mL centrifuge tube.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
LB Broth (Miller)	EMD Millipore	1.10285.0500
Tryptone, Poweder	Research Products International	T60060-1000.0
Yeast Extract, Poweder	Research Products International	Y20020-1000.0
Anhydrous magnesium sulfate	P212121	CI-06808-1KG
Sodium Chloride (Crystalline/Certified ACS)	Fisher Scientific	S271-10
Potassium Chloride	Fisher Scientific	BP366-500
Elga PURELAB Flex 3 Water Purification System	Fisher Scientific	4474524
Potassium Phosphate Monobasic	Fisher Scientific	BP362-1
Potassium Phosphate Dibasic Anhydrous	Fisher Scientific	P288-500
Isopropyl β-D-1-thiogalactopyranoside (IPTG)	Sigma Aldrich	I6758-1G
Nalgene Centrifuge Bottles with Caps, Polypropylene Copolymer	Thermo Scientific	3141-0250
VWR Os-500 Orbital Shaker	VWR Scientifc Products	14005-830
P1000 Pipetman	Gilson	F123602
Falcon 50 mL Conical Centrifuge Tube	Fisher Scientific	14-432-22