TITLE: Protein expression and extraction of hard-to-produce proteins in the periplasmic space of Escherichia coli
AUTHORS: Cristina Hernandez Rollan, Kristoffer Bach Falkenberg, Maja Rennig, Andreas Birk Bertelsen, Morten Norholm

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-style:italic;">E. coli </span><span>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.</span></div><div class = "text-block"><span>This protocol describes how to express proteins in the periplasm </span><span style = "font-style:italic;">E. coli </span><span>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 </span><span style = "font-style:italic;">E. coli</span><span>, as for instance, LPMOs.</span></div><div class = "text-block"><span>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. </span><span style = "font-style:italic;">Nat. Chem. Biol.10</span><span>, 122–126.</span></div><div class = "text-block">.</div></div>

[STEPS]
?. [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  at 250 RPM shaking . 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
37 °C
?. [Inoculation, Induction and expression - Day 2]
Dilute the overnight culture 1:100 in fresh LB supplemented with relevant antibiotics
?. [Harvesting and periplasmic extraction - Day 3]
Incubate the suspension at  for
25 Room temperature
?. [Harvesting and periplasmic extraction - Day 3]
Cold-shock the cells by adding  of ice-cold sterile MQ water per gram of cells (or alternatively,   of ice-cold water per every OD600 Unit can be added).
3 mL
12 µl
?. [Harvesting and periplasmic extraction - Day 3]
Spin down the cells at and collect the supernatant
Centrifuge: 8000 34, 20 min, 4 10
The supernatant contains the periplasmic extraction
?. [Inoculation, Induction and expression - Day 2]
Move the culture into an incubator set to   with 180 RPM of shaking and grow the culture to OD600 = 0.8 - 1.0
18 °C
?. [Inoculation, Induction and expression - Day 2]
Induce the expression by adding IPTG to a final concentration of
?. [Inoculation, Induction and expression - Day 2]
Let the culture grow at   with 180 RPM shaking for
18 °C
Expression at low temperatures is recommended to enhance the solubility of some proteins.
?. [Inoculation, Induction and expression - Day 2]
Grow the culture at  with 250 RPM shaking until an OD600 = 0.5 - 0.6
37 °C
?. [Harvesting and periplasmic extraction - Day 3]
Spin the culture down at  and discard the supernatant
Centrifuge: 8000 34, 20 min, 4 10
Remove as much of the remaining liquid as possible from the centrifuge tube. This helps greatly in periplasmic extraction.
?. [Harvesting and periplasmic extraction - Day 3]
Incubate the suspension  for
on ice
?. [Harvesting and periplasmic extraction - Day 3]
Keep the  extraction  when working with it and at  for storage
on ice
4 °C
?. [Harvesting and periplasmic extraction - Day 3]
Resuspend the pellet in  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  TSE per OD Unit is added)
3 mL
12 µl
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.