This is a rapid method to check out new fusion protein expression constructs. It works equally well for GST fusions, MBP fusions, and His-tagged fusions. It is designed to allow you to test a number of constructs simultaneously. If expressing GST or MBP fusions, an excellent positive control to do in parallel with your construct is to use the GST or MBP fusion vector (with no insert) to express and purify GST or MBP: these proteins are extremely well expressed, soluble, and bind to the appropriate affinity resin very well. Following this procedure will give you a first approximation of how well your fusion protein is expressed, how soluble it is, and how well it binds the appropriate affinity resin. However, certain fusion proteins that are not successfully expressed under these conditions can be expressed successfully by optimizing the induction protocol (OD at which you induce, time of induction, temperature of growth, concentration of IPTG, etc.) so don't give up if you things don't work immediately.
Growth
1. Grow an overnight culture (2 mls) of each of the expression strains you are testing in L Broth plus ampicillin (75 µg/ml) at 37°, shaking.
2. Dilute 0.75 mls of each culture into 75 mls of L Broth plus ampicillin, and grow with shaking at 37°. Monitor the growth of the cultures starting after about 1.5 hrs by taking the OD550.
3. When a culture reaches OD550=0.4 (this will take about 2 hours, depending on the strain used) the culture is ready to induce. First take out a 1 ml aliquot to prepare a gel sample of the uninduced cells. Spin down the bacteria in a microfuge for 30 seconds, and resuspend the pellet in 20 µl of SDS sample buffer. (General rule for making gel samples of E. coli: resuspend 1 OD ml of cells in 50 µl of sample buffer. In this case you are using 0.4 OD mls, and thus use 20 µl of sample buffer).
4. Induce the culture by adding 180 µl of 0.1 M IPTG.
5. Allow the induced culture to grow for 2 hours, shaking at 37°.
6. At the end of the growth, take the OD550 of the culture. Make a gel sample of these induced cells, again using 50 µl of sample buffer per OD ml of cells.
Note: for C. elegans proteins we have switched from 37° growths to doing all growths at 25° C or below. Worm proteins may be unstable at temperatures above those at which worms have evolved to live, and empirically we find that our worm fusion proteins are much more often soluble at lower temperatures.
Extract
7. Spin down 50 mls of the culture (can use an Oak Ridge or disposable 50 ml centrifuge tube, spin at ~6K for 5 minutes).
8. Wash the cells: discard the supernatant, and resuspend the cells in 10 mls of PBS, transfer to a 15 ml disposable centrifuge tube, and spin the cells down again. Resuspend the pellet in 2.5 mls of PBS.
9. All subsequent steps should be done on ice or in the cold room, as rapidly as possible. If you want at this point you can add protease inhibitors to the cell suspension to minimize protein degradation. My usual cocktail is 0.5 mM PMSF, and leupeptin and pepstatin at 1 µg/ml each. PMSF is unstable in aqueous solution, so add it immediately before use, and then work quickly!
10. Lysis. Place the 15 ml tube such that a sonicator microtip is suspended with its tip well below the surface of the liquid in the tube. Place a beaker of ice water around the tube to keep it cold during sonication. Sonicate with the sonicator turned up to the limit for the microtip for 1 minute. Some sonicators allow you to sonicate in e.g. 1 sec bursts with 1 sec rest between to try to keep the sample from heating too much during sonication.
11. Spin the lysed cells hard for 20 minutes at 4° C to remove insoluble material and unlysed cells. This should preferably be done at 100,000 X G. This is conveniently done in a Beckman tabletop ultracentrifuge using the TLA 100.4 rotor at 35,000 rpm in hard-wall polycarbonate tubes.
12. Remove the supernatant - this is the soluble extract. Make a gel sample by mixing 50 µl with 50 µl of SDS sample buffer. Most of the remainder of the extract can be flash frozen in 500 µl or 1 ml aliquots for future testing. If you will immediately proceed with resin binding, keep 500 µl at 4° for this test.
13. Make a gel sample of the insoluble material. Resuspend the pellet from the hard spin in 2.5 ml of PBS (use a spatula to scrape the pellet out of the tube and then use a dounce homogenizer to resuspend it in the PBS). Then mix 50 µl of the suspension with 50 µl of sample buffer. This sample will make the insoluble material to the same dilution as the soluble material gel sample, so that if equal volumes of each sample are loaded on a gel they can be directly compared.
Analytical resin binding
14. Prepare 50 µl of packed affinity resin (Glutathion agarse, amylose resin, or nickel resin, as is appropriate for the affinity tag you are using. The resins are supplied at 50% slurries, so pipette 100 µl of slurry into a screw cap eppendorf tube. Spin the resin down (this and subsequent spins should be done for 30 seconds at 1000 RPM in a microfuge. You should use a swinging bucket rotor in the microfuge if possible - this packs the resin to the bottom of the tube instead of sticking to the side). Remove the supernatant, and wash the resin 3 times with 1 ml of PBS (i.e. resuspend the resin in PBS, spin down, remove the supernatant).
15. Binding. Add 500 µl of extract to 50 µl of washed resin, resuspend the resin, and allow to rotate (on a Labquake rotator) at room temperature for 5 minutes. Spin down the resin, and remove the supernant. Make a gel sample (the "flow though") of the supernatant by mixing 50 µl with 50 µl of SDS sample buffer. Discard the rest of the supernatant.
16. Wash the resin 3 times with 1 ml PBS.
17. Add 50 µl of SDS sample buffer to the washed resin.
18. You now have generate 6 protein samples:
1. uninduced cells
2. induced cells
3. soluble extract
4. insoluble material
5. Unbound material from batch purification ("flow through")
6. resin bound (purified) protein
Using a 0.75 mm thick SDS acrylamide gel with ~0.5 cm wide
lanes you should load 20 µl of each sample.
Solutions:
Protease inhibitor stock solutions:
Pepstatin is suspended to 10 mg/ml in EtOH, leupeptin is dissolved to 10 mg/ml in DMSO, and the two solutions are mixed 1:1 and stored frozen to make a 5,000X leu + pep stock.
PMSF is kept as 200 mM in isopropanol at room temp. Be careful: protease inhibitors are extremely toxic, especially PMSF.
PBS
20 mM sodium phosphate, pH 7.2, 150 mM NaCl
SDS sample buffer
100 mM Tris, pH 6.8, 2% SDS, 5% ß-mercaptoethanol, 15% glycerol, enough bromophenol blue to make it dark looking. Store in a tightly sealed container to keep the BME from going off. You may still need to add more BME if your solution is old, since it is highly volatile. Some people keep sample buffer -BME and add it right before use.