TITLE: Protein Purification for OnePot PURE cell-free system
AUTHORS: Konstantinos Ragios

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">In this protocol we explain the procedure of protein purification with a single coculture and a single Ni-NTA affinity His-tag purification to produce the protein solution needed for a PURE cell-free system.</div></div>

[STEPS]
?. [Small cell culture ]
Prepare 10ml of  autoclaved LB medium and supplement it with 100μg/ml Ampicillin
Work under flame Sterilize the bench with 70% Ethanol
?. [Small cell culture ]
Fill 35 of the wells of a 96 deep-well plate with 0.3 ml of the LB medium according to the pattern of the stored strains.
We assumed that the 35 different bacterial strains are stored as a glycerol stock in a 96-well plate and the bacteria that express the EF-tu are also stored as a glycerol stock
?. [Small cell culture ]
Add  3ml of the LB medium to a standard 14ml culture tube (needed for the EF-Tu culture)
If you plan to do 2 different OnePot Protein purification batches you need to prepare one more tube for the EF-Tu culture
?. [Small cell culture ]
Add an arbitrary volume of stored strain with EF-Tu encoding plasmid.  20-50μl should be sufficient.
?. [Small cell culture ]
Use a  96-well replicator to innoculate the deep-well plate. Disinfect the replicator very well, with ethanol and flame and then dip it first in the bacterial stock and then into the 96 deep-well plate.
If you are performing this experiment for the first time and you don't have a well plate with stored bacteria, add the strains you acquired to their respective well,add an arbitrary volume in a way that you don't alter the final volume of 0.3ml significantly but making sure that at least some bacteria are transferred to the new solution, and after you culture them (step x) you can use the overnight culture to create your stock solution by adding to them  40% glycerol stock with a ratio of 1:1 and store at -80οC.
?. [Small cell culture ]
Cover the plate with a Breath-Easy sealing membrane to ensure no sample is spilled but also maintaining aerobic culture conditions.
?. [Small cell culture ]
Incubate overnight (12-16 hours) at 37οC rotating at 260 rpm
?. [Large Cell culture ]
It is advised to produce two batches of protein solution at the same time. All you need to do is repeat all the following steps for the second batch. However, even if the procedure for both of them will be the same, using the same stock buffers, you should not mix the two solutions at any point of the purification. In that way if there is any problem with one of them, it will not affect to the other one.
?. [Large Cell culture ]
Add 500ml of autoclaved LB medium into a 1L baffled flask and supplement it with 100μg/ml Ampicillin
Work under flame Sterilize the bench with 70% Ethanol
?. [Large Cell culture ]
Inoculate the the LB with 1675μl from the overnight culture of the EF-Tu strain and 55μl from each of the rest 35 the cultures.
?. [Large Cell culture ]
Incubate for 2hours at 37οC rotating at 260 rpm
?. [Large Cell culture ]
Induct with 0.1mM IPTG  and continue incubating for 3 more hours
?. [Cell suspension and lysis]
Harvest the cells in two tubes by centrifugation at 4000rpm for 10minutes in 4οC
Precool the centrifuge at 4οC
At this point you can store the tubes at -80οC overnight
?. [Cell suspension and lysis]
Prepare 7.5 ml of Buffer A, by adding 7mM β-Mercaptethanol (usually the stock solution is 2000x)
?. [Cell suspension and lysis]
Resuspend the cells
?. [Cell suspension and lysis]
First use 5ml to resuspend the cells in the first tube, then transfer the solution to the second tube and resusend the cell. Finally use the other 2.5 ml of buffer to wash the first tube for any cells left behind and move the solution to the second tube.
?. [Cell suspension and lysis]
Sonicate the solution with probe tip of 6mm with 4 cycles of a 20s on - 20s off pulse at 70% amplitude.
Clean the sonicator by applying a working cycle to a tube with milli-Q water
Keep the tube in ice to avoid heating up the solution during the sonication process.
?. [Cell suspension and lysis]
Centrifuge the lysate at 15000rpm for 20minutes at 4ο C. The debris form a pellet on the wall of the tube.
Precool the centrifuge at 4ο C
?. [Ni-NTA Resin Preparation]
Pipette 2 ml of IMAC Sepharose 6 FF into an Econo-Pac chromatography column
?. [Ni-NTA Resin Preparation]
Charge with 15ml of 100mM Nickel sulfate solution
Instead of the IMAC Sepharose you may work with a ready-to-use Ni-NTA resin. In this case you need to skip this step.
?. [Ni-NTA Resin Preparation]
Wash with 50ml of DEMI Water
Do not let the resin dry out entirely at any point of the experiment.
?. [Ni-NTA Resin Preparation]
Equilibrate with 35ml of Buffer A ( don't forget to add the β-Mercaptoethanol).
?. [Protein Purification ]
Put the supernatant of step 14 into the column with the resin and incubate at  4οC rotating slowly for 3 hours.
?. [Protein Purification ]
Let the unbound lysate to flow through the column.
?. [Protein Purification ]
Wash with 25ml of wash buffer consisting of 95% Buffer A and 5% Buffer B
?. [Protein Purification ]
Elute the proteins using 5m of Elution buffer (10% Buffer A and 90% Buffer B) and collect the solution.
?. [Resin regeneration]
Regenerate the resin with a 10ml mixture of 0.2M EDTA and 0.5M NaCl
?. [Resin regeneration]
Wash with 30ml of 0.5M NaCL
?. [Resin regeneration]
Wash with 30ml of DEMI water
?. [Resin regeneration]
Store in 20% Ethanol at 4ο C.
The resin can be reused. Before the next experiment you need to let ethanol flow through and then wash with DEMI water before starting the new preparation.
?. [Buffer exchange and concentration]
Mix the 5ml of elution solution with 25ml of HT Buffer.
?. [Buffer exchange and concentration]
Load half the new solution to a 15ml Amicon Ultra filter (3 kDa) and centrifuge for 60minutes at 4000 rpm at 4ο C.
?. [Buffer exchange and concentration]
After that, load the other half of the solution to the same filter and centrifuge for one more hour.
The expected concentrated quantity left at this point is around 1ml
?. [Buffer exchange and concentration]
Dilute the concentrated sample with 10 ml of HT Buffer and concentrate to 1.5ml (another 1 hour of centrifuge)
?. [Buffer exchange and concentration]
Measure the exact quantity you have left and add Stock Buffer B with a ratio of 1:1
?. [Buffer exchange and concentration]
Concentrate the solution with a 0.5ml Amicon Ultra filter ( 3 kDA) by centrifugation at 14000rpm for 30min at 4ο C.
?. [Measurements and Storage ]
Mix 24μl of water with 1μl of the concentrated solution and vortex it.
?. [Measurements and Storage ]
The final concentration must be 12.25mg/mLIf it is lower concentrate for more minutes and if it is higher dilute with Stock Buffer B
?. [Measurements and Storage ]
Perform a nanodrop measurement and calculate the sample's concentration: Multiply by 25 and divide by 0.5 (Extinction coefficient)
?. [Measurements and Storage ]
The Protein solution is stored at -80ο C