Jul 04, 2025
Large scale EV-A71 2A protease mature and precursor expression setup and operation of Single Use Bubble Column Reactors: Litre-Scale Expression of Recombinant Proteins for Structural Biology and Drug Design (SBDD)
- Nathan Wright1
- 1University of Oxford
- Nathan Wright: ASAP Discovery Consortium
- ASAP Discovery
Protocol Citation: Nathan Wright 2025. Large scale EV-A71 2A protease mature and precursor expression setup and operation of Single Use Bubble Column Reactors: Litre-Scale Expression of Recombinant Proteins for Structural Biology and Drug Design (SBDD). protocols.io https://dx.doi.org/10.17504/protocols.io.14egnypbqv5d/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 20, 2025
Last Modified: July 04, 2025
Protocol Integer ID: 220627
Keywords: Single-Use, Bubble Column Reactor, protein production, SBDD, 2A protease, Picornaviridae, E. coli, enterovirus 2a protease variant, inactive coxsackievirus a71 2a protease with c110a mutation, type human coxsackievirus a16 2a protease, a71 2a protease, inactive coxsackievirus a71, type human coxsackievirus a16, scale expression of recombinant protein, recombinant protein production, protein production methodology for structural biology, recombinant protein, protein production methodology, 2a protease, antiviral drug development, sufficient protein quantities for crystallographic study, protein, use bubble column reactor, bubble column reactor, providing sufficient protein quantity, magnetic bead purification, producing wet cell weight, purification, precursor expression setup
Funders Acknowledgements:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: U19AI171399
Disclaimer
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Abstract
This protocol describes a single-use Bubble Column Reactor (suBCR) system for large-scale recombinant protein production in E. coli, addressing limitations of traditional shake-flask methods for structure-based drug discovery. The system enables parallel 1-liter batch cultivation using disposable bioreactor bags in a heated water bath with controlled aeration.
Two enterovirus 2A protease variants were successfully expressed: wild-type Human Coxsackievirus A16 2A protease (Addgene 228632) and an inactive Coxsackievirus A71 2A protease with C110A mutation (Addgene 228633).The C110A mutation preserves the VP1-2A junction while eliminating catalytic activity, providing active and inactive variants for comparative studies that are suitable for crystallography. Cultures are grown at 37°C for 4 hours until exponential phase, then expressed overnight at ambient temperature (25-27°C) using auto-induction Terrific Broth media. The system achieved high yields with culture volumes of 5-22.6 L producing wet cell weights of 163.2-225.6 g. Quality control via nickel-magnetic bead purification and SDS-PAGE confirmed successful overexpression of both constructs.
This scalable, cost-effective approach significantly advances protein production methodology for structural biology and drug discovery, offering increased throughput and reduced labor compared to conventional methods while providing sufficient protein quantities for crystallographic studies and antiviral drug development.
Attachments
PAGE24-00929 (1).pdf
1.1MB
Materials
Media (auto-induction):
Formedium AIMTB0310 https://formedium.com/product/aim-terrific-broth-base-including-trace-elements/
Lysis reagent: FastBreak (V857C or V8571) from Promega (https://www.promega.co.uk/)
Protocol materials
Human Coxsackievirus A16 strain G10 2A proteaseaddgeneCatalog #228632
Enterovirus Coxsackievirus A71 inactive 2A protease with mutation C110A to preserve VP1-2A junction.addgeneCatalog #228633
Troubleshooting
Before start
The reader should be familiar with the companion protocol: "The Manufacture and Setup of Single Use (SUB) Bubble Column Reactors (BCR): Litre-Scale Expression of Recombinant Proteins for Structural Biology and Drug Design (SBDD)."
Culture Main Bags
4h 32m
Bag setup: a) Guidance for Bag manufacture and assembly, b)
Arrangement of the Bags over the hanging rail, c) A Bag Array in operation.
Bag set-up a) Bags removed from water bath for illustrative purposes, b) a
typical bubble swarm and foam layer, c) close-up of the Bags in the water bath
with heaters, d) a heat map showing water bath heating of the Bags;
left-to-right Bags acclimating.
Prepare and autoclaved Terrific Broth Base including Trace elements (Formedium AIMTB310)
To each litre of sterilised media:
Add1 mL 10% Antifoam 204
Add1 mL 1000x antibiotic stock (e.g. Kanamycin 50mg/ml)
Add20 g glycerol
Equilibrate the incubator bath:
Start the immersion heater program 37 °C 06:00:00
Allow incubator to reach set temperature.
Loading and equilibrating the BCR bags:
Add 1 L prepared AIM TB media to each BCR bag.
The bag with airline inserted, and a funnel being used to demonstrate the filling process.
Transfer BCR bags to incubator.
Insert sparger stones into BCR bag, making sure they are at the bottom of the BCR.
Attach sparger stones to air control valves. Set air flow, 1 lpm.
Allow bags to equilibrate.
Inoculate Media:
Note
If cultures are innoculated by late morning biomass growth can be monitored in person. If choosing to manually induce protein expression by IPTG this can be done before the end of the work day.
Expression can run unattended overnight.
Add 10 mL starter culture, per L main culture.
Biomass Growth Phase:
Continue incubation 37 °C , 04:00:00 (approximately).
Note
Incubation at 37 °C should continue until the culture has entered the exponential growth phase. Typically this takes 4-6 hours given the conditions described. Autoinduction media will trigger protein expression once all the glucose in the media has been consumed, this happens at OD600 of between 3-5 in most cases. The incubation temperature should be reduced before the autoinduction point to promote protein solubility.
4h
Protein expression phase
Protein Expression Phase we used the plasmid Human Coxsackievirus A16 strain G10 2A proteaseaddgeneCatalog #228632 andEnterovirus Coxsackievirus A71 inactive 2A protease with mutation C110A to preserve VP1-2A junction.addgeneCatalog #228633
Incubate (ambient)Overnight (approximately 16 h)
- [EndPoint] ΔOD600≈0
Note
Without external cooling cultures' metabolic activity will heat the culture to 25-27 °C . If working with proteins with temperature sensitive expression, an external recirculating chiller can be connected to the water bath.
Note
- A typical culture, having been innoculated at between 10-12 pm on day 1, can be harvested in the morning of day 2. This gives a total culture time of 20-24 hours.
- Culture times of up to 36 hours have been tried successfully, but this is likely to be cell/protein specific.
- Short expression experiments can be performed with a c.3 hour induction period following the addition of IPTG.
- Culture biomass can be measured by taking Optical Density readings at 600 nm in a spectrophotometer. The relationship between OD600 and biomass is non-linear at high concentrations. If using a fixed path-length or cuvette, dilute samples such that readings remain below 1 OD unit.
- If using autoinduction media and cell growth appears to have stalled below OD600=5, IPTG can be added to ensure induction has occurred.
Note
- In rich media biomass will continue to increase after induction.
- Once OD600 values plateau late log phase has occurred, and cell harvest must occur before OD600 begins to drop when death phase begins.
Harvest
Harvest:
Take final OD600 reading
Collect 1 mL culture samples for diagnostic testing
Transfer culture to 1 L centrifuge bottles
Note
The Bags are long and flexible, decanting by pouring culture out from the top of bags can lead to spillage.
Place Bags into a large jug or similar. With a sharp implement, make a small incision in the bag and allow it to drain into the jug.
There is a lot of hydrostatic pressure on the liquid at the bottom of the Bags when full. To avoid splashing make incisions towards the top of the liquid level at first. As the culture drains, larger incisions can be made at the bottom to drain all liquid and sediments.
Pellet the cell, 4000 rcf, 4°C, 00:30:00
30m
Drain spent media and sterilise with 1 tablet/L Virkon
Transfer cell pellet to cryo-safe sample bags. Mark with full and clear tracking information.
Record wet cell weight (g)
Flash freeze the cell pellet in liquid nitrogen.
Store cell pellet -80 °C
Overexpression quality control
Overexpression QC:
Note
Using Nickel Magnetic Beads perform a rapid over-expression assay. The protocol described here is abridged and achieves only incomplete cell lysis, but can be used to varify over-expression in 5-10 minutes, plus gel running time.
Transfer 1 ml of culture sample to a microcentrifuge tube
Add 100 µL lysis reagent to the sample
Note
If biomass is low or protein expression levels are expected to be low, take a larger culture sample and pellet it first. This will increase the target protein yield but you may need to check sufficient lysis has occurred.
Incubate Room temperature 00:02:00 in a tube rotor
Pellet the cell debris at maximum speed in a microtube centrifuge 16000 rpm, 00:02:00
Note
There is no need to spin at 4 °C unless you plan to use the eluted protein for other purposes than QC SDS-PAGE.
Aspirate soluble fraction (mixed proteins) with pipette
Add 50 µL magnetic IMAC beads to the soluble fraction (mixed proteins)
Incubate Room temperature 00:05:00 in a tube rotor
Incubate Room temperature 00:01:00 in a magnetic separator stand
Aspirate soluble fraction (unbound material), reserve for SDS-PAGE analysis.
Wash the retained beads 2 mL binding buffer 00:01:00
Incubate Room temperature 00:01:00 in a magnetic separator stand
Aspirate soluble fraction (unbound material), reserve for SDS-PAGE analysis.
Add 50 µL elution buffer to the mag beads
2m
Incubate 00:02:00 Room temperature
Incubate 00:01:00 in a magnetic separator stand
Aspirate soluble fraction (eluted protein) with pipette
Measure the A280 using a spectrophotometer (e.g. NanoDrop)
Run an SDS-PAGE gel, to check for over-expression and quality control
Results
Tracking IDs and Yields:
| A | B | C | D | E | F | G | H | I | J | |
| ELN title | Target ID | Clone ID | Construct ID | Expression ID | Culture Total Volume (L) | OD600 | Pellet Wet Cell Weight (g) | SDS-Page: Test purification Mag Prep (pass) | Expression Growth (Pass) | |
| Expression only A712A-c012 | A71EV2A | A71EV2A-k005 | A71EV2A-c012 | A71EV2A-e078 | 12 | 22.6 | 225.6 | Pass | Pass | |
| A71EV2A-c058 | A71EV2A | A71EV2A-k030 | A71EV2A-c058 | A71EV2A-e067 | 5 | 18 | 163.2 | Pass | Pass |
Annotated SDS-PAGE gel of Mag Prep test purification