Oct 27, 2020

Public workspaceProtein expression and extraction of hard-to-produce proteins in the periplasmic space of Escherichia coli

DOI
dx.doi.org/10.17504/protocols.io.bdr2i58e
  • 1Technical University of Denmark
Cristina Hernandez Rollan
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DOI: dx.doi.org/10.17504/protocols.io.bdr2i58e
Protocol Citation: Cristina Hernandez Rollan, Kristoffer Bach Falkenberg, Maja Rennig, Andreas Birk Bertelsen, Morten Norholm 2020. Protein expression and extraction of hard-to-produce proteins in the periplasmic space of Escherichia coli. protocols.io https://dx.doi.org/10.17504/protocols.io.bdr2i58e
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: March 16, 2020
Last Modified: October 28, 2020
Protocol Integer ID: 34330
Keywords: LyGo, LPMO, Periplasmic expression, E. coli, protein expression, Periplasmic extraction,
Abstract
E. coli is a gram-negative bacteria used mainly in academia and in some industrial scenarios, as a protein production workhorse. This is due to its ease of manipulation and the range of genetic tools available.

This protocol describes how to express proteins in the periplasm E. coli with the strain BL21 (DE3) using a T7 expression system. Specifically, it describes a series of steps and tips to express "hard-to-express" proteins in E. coli, as for instance, LPMOs.


The protocol is adapted from 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.
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Guidelines
The periplasm of E. coli is often the preferred strategy to produce heterologous proteins in this bacterium as it provides the means for disulfide bond formation.
The choice of the signal peptide is of great importance to ensure correct and efficient translocation to the periplasm. In our lab, we routinely screen five different signal peptides: MalESP, OmpASP, PhoASP, DsBASP, and PelBSP.
Materials
MATERIALS
ReagentBL21(DE3) Competent E.coli - 6x0.2 mlNew England BiolabsCatalog #C2527I
ReagentIPTGBio Basic Inc.Catalog #IB0168.SIZE.100g
ReagentEDTAFisher ScientificCatalog #16 004Y
ReagentKanamycinResearch Products International Corp (RPI)Catalog #K22000-25.0
ReagentLBResearch Products International Corp (RPI)Catalog #L24400-2000.0
ReagentSucroseFisher ScientificCatalog #S25590B
ReagentTrizma® baseMerck MilliporeSigma (Sigma-Aldrich)Catalog #93362
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.
Pre culture - Day 1
Pre culture - Day 1
Pick a fresh colony of your BL21 (DE3) strain with your expression vector, and inoculate it in LB supplemented with relevant antibiotics. Grow the culture at Temperature37 °C at 250 RPM shaking DurationOvernight . 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

Inoculation, Induction and expression - Day 2
Inoculation, Induction and expression - Day 2
Dilute the overnight culture 1:100 in fresh LB supplemented with relevant antibiotics
Grow the culture at Temperature37 °C with 250 RPM shaking until an OD600 = 0.5 - 0.6


Move the culture into an incubator set to Temperature18 °C with 180 RPM of shaking and grow the culture to OD600 = 0.8 - 1.0
Induce the expression by adding IPTG to a final concentration of Concentration1 millimolar (mM)

Let the culture grow at Temperature18 °C with 180 RPM shaking for Duration20:00:00
Note
Expression at low temperatures is recommended to enhance the solubility of some proteins.


Harvesting and periplasmic extraction - Day 3
Harvesting and periplasmic extraction - Day 3
10m
10m
Spin the culture down at Centrifigation8000 x g, 4°C, 00:20:00 and discard the supernatant

Note
Remove as much of the remaining liquid as possible from the centrifuge tube. This helps greatly in periplasmic extraction.

Resuspend the pellet in Amount3 mL of buffer TSE buffer (200 mM Tris-HCl pH 8, 500mM sucrose, 1mM EDTA) per gram of cells (this normalization can also be based on OD600 where Amount12 µL TSE per OD Unit is added)

Note
Carefully resuspend the cells in the TSE buffer to avoid breaking the cells. A good tip is to use a sterile inoculation plastic loop to resuspend the pellet in the buffer before using a pipette tip.

Incubate the suspension at TemperatureRoom temperature for Duration00:10:00

10m
Cold-shock the cells by adding Amount3 mL of ice-cold sterile MQ water per gram of cells (or alternatively, Amount12 µL of ice-cold water per every OD600 Unit can be added).


Incubate the suspension TemperatureOn ice for Duration00:10:00

Spin down the cells at Centrifigation8000 x g, 4°C, 00:20:00 and collect the supernatant
Note
The supernatant contains the periplasmic extraction

Keep the extraction TemperatureOn ice when working with it and at Temperature4 °C for storage