This article details a protocol for preparing crude cell extracts from Vibrio natriegens, known for its rapid protein synthesis. The method emphasizes maintaining cold conditions to preserve cellular integrity and bioactivity during extraction.
Take a suspension of Vibrio natriegens, which has a high protein synthesis rate, in a chilled buffer to prevent cellular degradation.
Centrifuge under cold conditions.
Discard the supernatant to remove contaminants.
Resuspend the pellet in a small volume of chilled buffer.
Using a wide-bore tip, transfer the suspension into a microcentrifuge tube to prevent mechanical shearing.
Mix the suspension and place the tube in a holder positioned on ice.
Lower the sonicator tip into the suspension.
Initiate sonication to disrupt the cell membrane and release cytoplasmic contents.
Centrifuge to separate the insoluble cell debris.
Collect the supernatant or crude cell extract containing cytoplasmic contents into a fresh tube.
Flash freeze the cell extract in liquid nitrogen to preserve its bioactivity.
Store at ultra-low temperatures for further use in cell-free protein expression.
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 litre 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 one-eighth-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.
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