This protocol for a recombinant cell-free protein expression system, commonly known as the PURE system utilizes cell culturing and cell purification to streamline the required protein purifications. This significantly minimizes the time and labor requirements. The OnePot method has proved to be not only cost-effective, but also time efficient.
It cuts down the preparation times from several weeks to three working days. This makes the PURE cell-free platform accessible to more laboratories worldwide and helps to implement this really powerful platform for synthetic cell-free biology. Before you start, ensure that all strains over express their respective protein well.
To begin, prepare the column for the OnePot protein purification as described in the text manuscript using two milliliters of Sepharose resin, and then charge the column with nickel sulfate. Prepare the starter cultures by combining 20 milliliters of LB media with 20 microliters of ampicillin. Add 300 microliters of the media into 35 wells of a sterile 96 deep well plate.
Inoculate each of them with its respective strain, except elongation factor thermo unstable, or EFTU, and seal the plate with a breathable membrane. For the EFTU culture, inoculate three milliliters of ampicillin containing LB media in a 14 milliliter culture tube with a snap cap. About three milliliters of EFTU culture is sufficient for one OnePot expression culture.
Incubate it at 37 degrees Celsius while shaking at 260 rotations per minute overnight. The next day, transfer 500 milliliters of LB media and 500 microliters of ampicillin into the sterile baffled flask. Inoculate the OnePot PURE culture with 1, 675 microliters of the EFTU culture and 55 microliters of each of the cultures from the deep well plate.
Incubate the culture for two hours at 37 degrees Celsius with a shaking of 260 rotations per minute, or until the optical density of the culture reaches 0.2 to 0.3. Induce the culture with 500 microliters of 0.1 millimolar IPTG and grow for an additional three hours. Harvest the cells by centrifugation at four degrees Celsius and 3, 220 times G for 10 minutes, and store the cell pellet at minus 80 degrees Celsius until further use.
On day three, measure the amounts of buffers needed for their purification and add TCEP to all of them as indicated in the text. Store the remaining buffers without TCEP at four degrees Celsius for future purifications. Equilibrate the charged column with 30 milliliters of buffer A.After 25 milliliters of buffer A has passed through, close the column from the bottom.
Thaw the cells and use a serological pipette to resuspend the cell pellet in 7.5 milliliters of buffer A.Lyse the cells using a 130 watt probe. If the sonication is successful, the solution will turn darker. Make sure to keep the cells on ice and place the probe deep enough without touching the tube.
Remove the cell debris by centrifugation at 21, 130 times G for 20 minutes at four degrees Celsius and keep the lysate on ice. Add the supernatant to the equilibrated column. Close the column from the top and make sure there is no leakage.
Incubate the column for three hours at four degrees Celsius under rotation using a tube rotator. Elute unbound components from the column and wash with 25 milliliters of buffer A.Wash the column with 25 milliliters of 25 millimolar imidazole buffer. Elute the proteins with five milliliters of 450 millimolar imidazole buffer and keep the eluted proteins on ice at all times.
Dilute the eluate with 25 milliliters of HT buffer and keep the mixture on ice. Add 15 milliliters to a 15 milliliter centrifugal filter and concentrate to a volume of 1.5 milliliters. Add the remaining 15 milliliters to the filter with the concentrated solution and concentrate to 1.5 milliliters once more.
Add 10 milliliters of HT buffer to the concentrated sample and concentrate to one milliliter. Recover the concentrated sample and add an equal amount of stock buffer B and store at minus 80 degrees Celsius until further use. During the buffer exchange, restore the column as specified in the text.
On day four, measure the protein concentration using the Bradford assay. Concentrate the sample with 0.5 milliliters of three kilodalton cutoff centrifugal filter to 20 milligrams per milliliter. Verify the OnePot PURE protein composition using SDS page.
Dilute 2.5 microliters of the sample with 7.5 microliters of water mixed with 10 microliters of two X Laemmli buffer, and then load five microliters and 2.5 microliters of the samples on the gel. Run the SDS page as specified in the text. Fill in the concentration and length of the DNA in the corresponding yellow cells in the spreadsheet using two to 10 nanomolar of DNA for the reaction.
Fill in the desired total reaction volume in microliters. Remove the required reagents from the freezer and thaw them on ice. Then, pipette the calculated amounts of water, DNA and energy solution to one side of the PCR tube or one corner of a well on the 384 well plate.
Pipette the calculated amounts of protein and ribosome solution to the other side of a PCR tube or the opposite corner of the 384 well plate. Spin for about 30 seconds to merge the reaction components. To prevent evaporation during plate reader experiments, add 35 microliters of liquid wax, and seal the plate with a transparent sealant.
Incubate for a minimum of three hours at 37 degrees Celsius. For readout on a plate reader, measure the florescence intensity at the required wavelength every two minutes. Successful over expression in all individual strains used subsequently for the OnePot protein preparation is shown here.
The overall composition of the final OnePot PURE protein solution was analyzed by SDS page. It is noticeable that EFTU is present in a higher concentration compared to the other proteins, as expected. The synthesis yields of the OnePot protein solution combined with the His-tagged or non-tagged ribosomes were analyzed by monitoring the eGFP fluorescence over time.
The expression levels of three different proteins labeled using the green list tRNA in vitro labeling system, or Kumasi staining, were analyzed on an SDS page gel. For a functional PURE system, it is absolutely critical that all strains express their respective proteins well. This technique facilitates the implementation of the PURE system to biological system engineering, enabling the development of novel genetic networks, molecular diagnostics, therapeutics and educational kits.
