For the BL21 cells, there are three potential methods for lysis:
The BL21(DE3)pLysS cells have been engineered to constitutively express T7 lysozyme when under chloramphenicol pressure as the pLysS plasmid contains the gene for the lysozyme. The main function of the T7 lysozyme is RNA polymerase inhibition, thereby preventing any “leaky” expression of the pET derived gene. Leaky expression can be a problem if the recombinant protein is toxic. However, there is no evidence that this would be the case for the nitrilases. Therefore, to take advantage of the freeze-thaw, cells are repeatedly frozen (fairly slowly in liquid nitrogen) and thawed on ice (around 3-5 cycles). This results in damage to the inner membranes of the cells and the T7 lysozyme leaks out of the cytoplasm and begins to digest the outer cell wall of the bacterium, thereby lysing the cells. This sets off a chain reaction with each lysed cell releasing lysozyme to digest other cell walls. This method for lysis is not used in the case of nitrilases as it has been noticed that purified nitrilases suffer structural damage upon freezing.
2. Lysozyme digestion
With this method, hen egg white lysozyme (muramidase) is added to the cell suspension and lysis allowed to continue for 30 min – 1 hour. The enzyme is provided as a powder and is usually kept in the walk-in fridge on the 2nd floor in MCB. If you want to buy your own stash of lysozyme, get it from Roche as they are the cheapest and quickest. If you use the pool lysozyme in MCB, be sure to check the expiry date on the bottle. Lysozyme is a highly cationic protein with an isoelectric point around 11.0. Therefore, although it is used at high concentration for bacterial lysis (1 mg/ml final concentration), it will not bind the Q-Sepharose anion exchanger and contaminate the protein purification. Most methods available carry out lysozyme digestion on ice, however, if your protein is fairly thermostable, I would recommend performing the digestion at 25-30°C as this is the optimal temperature for lysozyme. Keeping the temperature optimal is an important consideration as lysozyme will be digesting E. coli at a pH far more acidic than its optimum (pH 9.2).
For lysozyme digestion:
- Dissolve lysozyme in an appropriate amount of buffer (same buffer as the one used for pellet reconstitution; water can also do for lysozyme) to make a 10 mg/ml solution. Add the enzyme powder to the buffer and allow to dissolve slowly – do not shake or mix. Keep on ice.
- Resuspend E. coli pellet in an appropriate amount of buffer and add the lysozyme stock solution to give a final concentration of 1 mg/ml.
- Transfer the solution to the 30°C incubator and shake gently for 30 min-1 hour. At this point, if it is late in the day, one could then transfer the solution to the refrigerator and allow to continue digestion until morning.
- The resultant solution will be highly viscous due to the presence of genomic DNA. The viscosity can be reduced by passage through a 21 gauge needle (3-5 passages) or one could add MgCl2 and DNAse I. If performing DNAse digestion, this would probably have to be ordered in advance and you would follow the instructions on the package.
- Once the viscosity has been reduced, pellet the cell debris by centrifugation at 20 000 x g, 30 min, 4°C. Use the supernatant for the next step, i.e., salt precipitation or ion exchange.
This method is the most efficient and fairly quick. In MCB, there are 2 sonicators. A high-powered Virtis and a lower-powered MSE. The virtis has been away for repairs for many months, and so the method below has been optimised for the MSE. A major consideration when ultrasonicating is sample heating. The cavitation caused by the ultrasonicator can raise the temperature quickly, and the solution may get very hot in the vicinity of the tip. For this reason, one can do many short pulses rather than one long continuous pulse. Also, it is important to make sure that the cell suspension is chilled and that it is packed with crushed ice during sonication.
It is recommended that you read the paper: Optimised released of recombinant proteins by ultrasonication of E. coli cells by Feliu et al. [cite source=pubmed]10099290[/cite]. It describes how the authors optimised various parameters (time, sample volume, cell concentration, ionic strength, acoustic power) for release of recombinant 2-galactosidase. They found, unsurprisingly, that increased power and reduced sample volume increased recovery, but that cell concentration and buffer ionic strength had zero effect.
For the MSE sonicator, I have found the following to be optimal:
- Resuspend cell pellet in buffer (try to aim for a maximum of 10-20 ml) and add lysozyme to 1 mg/ml. Incubate at 30°C for 1 hour. Transfer the tube to crushed ice and allow to cool for 30 mins. This lysozyme pre-step is necessary as the power output from the MSE sonicator does not result in highly efficient lysis. The addition of lysozyme before sonication will help to “weaken” the cell walls.
- Sonicate for 2 min per 10 ml at 22 μm amplitude. You will notice that the tuning function on the machine is not operational. Make sure that the tip is inserted into the solution to a depth of at least 1 cm. Also make sure that the tip is not touching the bottom of the tube. Repeat the sonication until you notice the solution change colour from very milky to slightly more translucent. You may need to sonicate for a number of 2 min bursts, depending on the cell pellet mass. As the energy of the sonicator is fairly low, very little heating occurs. However, check the tube often and feel if it has warmed. Alternately, measure the liquid temperature with a thermometer that has been wiped with 70% ethanol.
- If you are STILL unsure, rather transfer the solution to the fridge and allow the lysozyme digestion to continue overnight.
- Pellet the cell debris by centrifugation at 20 000 x g, 30 min, 4°C.
(For cleaning of the tip after use, use 100% ethanol after switching off the machine)
If the Virtis is back in action, then rather use it than the MSE. However, you will have to monitor the sample very carefully as it heats up fast. You do not have to add any lysozyme if using the Virtis. Follow these steps:
- First, you will have to tune the tip. Assemble the ½ inch horn and tighten with the spanner. Read the instructions for tuning and ask Blommie or someone else if you are unsure. Wear the ear-muffs while tuning and sonication unless you think hearing aids are going to be the new fashion for under 30s. Make sure that the tip is inserted into the solution to a depth of at least 1 cm. Also make sure that the tip is not touching the bottom of the tube.
- Use power level 6 and pulse for 5 sec with a wait/delay for 1 min. Set the total time for 3 min. Although you will have programmed in a delay, pause the programme often to check the temperature of the solution and pack more ice around the tube if necessary.
- Switch off the machine and clean the ½ inch horn with 100% ethanol.
- Pellet the cell debris by centrifugation at 20 000 x g, 30 min, 4°C.