Our protocol for cell-free protein expression utilizes highly active crude extract from the fast growing bacterium Vibrio natriegens, which is prepared using a simple and time-efficient two-step sonication-centrifugation process. Crude cell extract preparation and cell-free protein synthesis can be achieved in one to two days by a single user allowing this technique to be easily integrated into pipelines for bioproduction or synthetic biology applications. To begin wash one milliliter of the overnight Vibrio natriegens culture by centrifuging it in a benchtop centrifuge at 10, 600 times g for one minute.
Aspirate the supernatant without disturbing the pellet, and resuspend in one milliliter of fresh LB-V2 media. Inoculate one milliliter of washed overnight culture into one liter of fresh LB-V2 growth media in an autoclaved four-liter baffled Erlenmeyer flask with sterile cover to achieve one-to-one thousand dilution ration. Grow culture at 30 degrees Celsius while shaking to 225 RPM.
Use a spectrophotometer to monitor the cultures OD 600. And when it reaches 1.0 plus or minus 0.2 harvest the culture by centrifuging it in two 500-milliliter tubes at 3, 500 times g for 20 minutes at four degrees Celsius, and then place on ice. Aspirate the supernatant and process the pellet immediately.
Before starting the cell lysis procedure cool freshly prepared S30 lysis buffer pH 7.7 to approximately four degrees Celsius, Use 10 milliliters of this cold S30 lysis buffer to re-suspend all pellets resulting from the same one liter culture. And transfer suspension to a 50-milliliter tube. Centrifuge this suspension at 3, 500 times g for 10 minutes at four degrees Celsius, and then aspirate the supernatant without disturbing the pellet and place it on ice.
Working in a cold room add 500 microliters of cold S30 lysis buffer to the pellet in the 50-milliliter tube placed on ice. Use a wide bore pipette tip to resuspend the pellet and carefully transfer it to a two-milliliter tube also on ice. In order to produce very active crude extracts we must make sure that the cells are not over diluted with S30 buffer, but have enough liquid for efficient sonication.
Fill a 600-milliliter beaker with ice and place a two-milliliter tube holder on top of the ice. Vortex the tube briefly to homogenize the cells. Flick to remove any cells on the bottom of the cap, and place into tube holder with cap open.
Lower sonicator tip into the cell suspension in the tube so that it is just under the liquid surface. Prepare the sonication set-up using a sonicator and probe with a 1/8-inch tip diameter. Set the sonicator at 20 kilohertz frequency and 50%amplitude.
Pulse-on time of 10 seconds, and pulse-off time of 60 seconds. Run the pulse sonication protocol for three cycles. And then centrifuge crude cell extract at 16, 000 times g for 30 to 45 minutes at four degrees Celsius until lysate is free of any cellular debris.
Visually inspecting cell lysis as sonication takes place is critical to ensure that sonication has worked as expected. Working in a cold room aliquot 50 microliters of the resulting supernatants to new two-milliliter tubes making sure not to disturb the pellet. To flash freeze these crude cell extracts place the tubes into a tube holder with a dipping string attached and submerge into a dewar containing liquid nitrogen and check if frozen.
To perform cell-free protein expression using DNA template thaw 10X energy solution master mix, 4X amino acid master mix aliquots, and DNA template at room temperature. Then remove the T7 RNA polymerase and RNAse inhibitor stocks from the minus 20 degrees Celsius freezer and place on ice. Once the DNA template, 10X energy solution master mix, and 4X amino acid master mix are thawed, place on ice.
Prepare a cell-free reaction master mix by adding each component in the accurate order to a two-milliliter tube on ice, and gently flick the tube after each addition to the master mix. Remove Vibrio natriegens crude cell lysate from the minus 80 degrees Celsius freezer and place on ice for 10 to 20 minutes until thawed. Add the appropriate volume of thawed crude cell extract to the cell-free reaction master mix and gently mix using a pipette.
To perform endpoint cell-free protein expression using thermocycler, first add 10 microliters of the cell-free reaction master mix per well of the bottom of a 96-well PCR plate making sure to mix the master mix by flicking the tube gently in between each transfer to the PCR plate. Centrifuge the plate at 1, 000 times g for 10 seconds to pool any master mix that may have been stuck to the sides of the wells. And then seal the wells with a plate adhesive to prevent evaporation.
Place the plate into a thermocycler set at 26 degrees Celsius with a heated lid set at 105 degrees Celsius. After incubating the reactions in the PCR plate for a minimum of three hours, expressed proteins can be purified, quantified and used for downstream processes. To monitor cell-free protein expression kinetics using a plate reader pipette 10 microliters per well of the cell-free reaction master mix to the bottom of a black 384-well assay plate with clear glass bottoms.
And seal the wells with a clear plate adhesive to prevent evaporation. Place the assay plate into a plate reader set at 26 degrees Celsius and incubate for three to six hours while monitoring the appropriate fluorescent excitation/emission wavelengths corresponding to the expressed protein. Cell-free expression system described in this protocol achieve the best results with plasmid DNA template yielding significantly higher amount of protein than the same amount of linear DNA as shown by endpoint and kinetic assays.
mRNA generated by in-vitro transcription reactions could also be used, but higher amounts of mRNA are required. The yield of protein production was significantly affected by the concentration of magnesium and potassium ions. Under optimized conditions the optimal magnesium ion concentration was found to be 3.5 millimolar and for potassium ion, 80 millimolar.
Thus deviations from these optimal ion concentrations may result in a decreased capacity for this expression system to produce significant yields of proteins. It is important to keep all reagents on ice or work in a coldroom as much as possible. Additionally, proper and complete sonication is critical to the success of this protocol.
Following this protocol users will have the capacity to express proteins in a high-throughput format, which can be used to purify and screen for activity of many different proteins. This technique demonstrates the use of new organisms with unique properties for cell-free expression. Vibrio natriegens is a halophilic fast-growing bacterium that may offer unique conditions for expression of proteins or small molecules previously unexplored in established model organisms.
