Oct 27, 2020
Protein expression of hard-to-produce proteins in the cytoplasm of Escherichia coli
- Cristina Hernandez Rollan1,
- Kristoffer Bach Falkenberg1,
- Maja Rennig1,
- Andreas Birk Bertelsen1,
- Morten Norholm1
- 1Technical University of Denmark
Protocol Citation: Cristina Hernandez Rollan, Kristoffer Bach Falkenberg, Maja Rennig, Andreas Birk Bertelsen, Morten Norholm 2020. Protein expression of hard-to-produce proteins in the cytoplasm of Escherichia coli. protocols.io https://dx.doi.org/10.17504/protocols.io.bfbwjipe
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: April 20, 2020
Last Modified: October 28, 2020
Protocol Integer ID: 35926
Keywords: E. coli, T7, Protein production, Cytoplasmic expression, sHuffle strain, disulfide bond
Abstract
This protocol describes the expression and extraction of heterologously cytoplasmically expressed proteins in the bacterium E.coli, for T7 based expression systems.
Specifically, it describes how to produce "hard-to-produce", as for example, LPMOs.
It is based on a modified version of:
"IPTG Induction and Extraction of Proteins from Bacteria" by Swathi Arur and Sudhir Nayak, Schedl Lab. Washington University Genetics, St. Louis.
Hemsworth, G. R., Henrissat, B., Davies, G. J., and Walton, P. H. (2014) Discovery and characterization of a new family of lytic polysaccharide monooxygenases. Nat. Chem. Biol.10, 122–126.
The extraction of cytoplasmatic proteins is followed by a chemical lysis protocol, alternatively, sonication can also be employed.
Guidelines
Make sure to clone your favorite gene for expression in the appropriate expression vector.
Materials
MATERIALS
SHuffle T7 Competent E.coli - 6x0.05 mlNew England BiolabsCatalog #C3026H
IPTGBio Basic Inc.Catalog #IB0168.SIZE.100g
BL21(DE3) or BL21-Star(DE3) or Rosetta2(DE3) or etc for protein purification
KanamycinResearch Products International Corp (RPI)Catalog #K22000-25.0
LBResearch Products International Corp (RPI)Catalog #L24400-2000.0
Safety warnings
This protocol describes the construction of GMO classified organisms. Make sure that the local GMO and safety legislations are respected.
Before start
Prepare a fresh transformation of your expression vector in E. coli BL21 (DE3) cells or other appropriate strain (e.g. sHuffle strain, Rosetta, etc.)
Day 1 - Culture inoculation
Day 1 - Culture inoculation
Pick a fresh colony of your BL21 (DE3) strain previously transformed with your expression vector, and inoculate it in LB medium supplemented with relevant antibiotics. Grow the culture at 37 °C at 250 RPM shaking Overnight . The volume of the overnight culture depends on the volume of the expression culture and should be at least 1/100 of the expression culture
Day 2 - Induction
Day 2 - Induction
Dilute the overnight culture 1:100 in fresh LB supplemented with relevant antibiotics
Grow the culture at 37 °C with 250 RPM shaking until an OD600 = 0.5 - 0.6
Move the culture into an incubator set to 18 °C with 180 RPM of shaking and allow the culture to reach OD600 = 0.8 - 1.0
Induce the expression by adding IPTG to a final concentration of 1 millimolar (mM)
Grow the culture for 20:00:00 at 18 °C with 180 RPM shacking.
Note
The temperature can be lowered further allowing for a slower induction in order to increase the production of soluble product.
Day 3 - Collection and lysis
Day 3 - Collection and lysis
Collect induced samples by centrifugation at 8000 x g, 4°C, 00:20:00 and carefully discard the supernatant
Note
Pellets can be frozen here at -80 °C or -20 °C until protein extraction
Lyse the cells using a lysis protocol or a commercially available kit
Keep the extractions On ice when working with it and at 4 °C for storage