From Plasmid to Protein
Step 1: Transform appropriate DNA plasmid into BL21(DE3) E. coli cells. These cells must be competent. (Protocol for how to make competent cells.)
a) Take competent cell stock aliquot (about 100 mL) out of -80oC freezer. Place on ice to thaw about 5 minutes.Step 2: Make a starter culture for protein expression. Usually to about 250 to 500 mL of LB broth the antibiotic is added.
b) Take one mL of plasmid DNA (usually a pRSET B vector with your protein cloned inside DNA seq of pRSET B) and add to the thawed competent cells. Vortex briefly to mix, and let incubate on ice for at least 20 minutes. Longer incubation will not hurt.
c) Heat shock the competent cells at exactly 42oC for 1 minute. Place on ice.
d) Add heat shocked cells to 1 mL of LB broth in a sterile culture tube, but DO NOT ADD antibiotic. Shake at 37oC for at least 1 hour. Shaking for 3 hours doesn't seem to hurt.
e) Plate the cell culture onto LB agar plates (usually 100 mL of culture will do although it depends on the competency of the cells and the initial concentration of plasmid). Make sure the plates have the appropriate antibiotic (usually ampicillin at 100 mg/ml concentration). (Protocol for how to make LB Ampicillin Agar Plates) Incubate the plate at 37oC for 14 hours (overnight generally). Longer times of incubations will allow for satellite colonies to appear.
a) Generally, the antibiotic is ampicillin, which is added to the LB broth at 100 mg/ml final concentration. (Have a stock of ampicillin in the 4oC which is 1000x concentrated or 100 mg/ml in sterile distilled water to make things easier).Step 3: Make the big batch of bacteria culture for protein expression. (How many 1.5 L volumes should you grow up? Expect about 100 mgs of protein per liter, but that estimate is very dependent on the protein!)
b) Pick a colony or two from your plate using a sterile innoculation loop and add to the LB broth/ampicillin flask. Incubate in a shaker (250 RPM) at 37oC until the OD @ 600 nm is 0.5 to 1.5 OD. (Anywhere in this range is fine, however, extremely dense starter cultures>1.5 OD can cause problems in protein expression. Under these dense growth conditions the plasmid seems to be rejected or turned-off by BL21(DE3)s.)
c) Save your starter culture flask by placing in the 4oC overnight. This will slow down the bacterial growth enough, but prevent the problems dense cultures cause as listed above.
a) Equally divide your starter culture among several 1.5 L volumes of sterile LB broth in 2.8 L Fernbach culture flasks. (Good precision when equally dividing the starter will better control the timing when induction can occur.)Step 4: Lysis and sonication of the bacteria. There are many ways to lyse bacteria, skin chickens etc., but this method is tried and true.
b) Add antibiotic to 100 mg/ml final concentration. Grow them up in a New Brunswick shaker large enough to hold several Fernbach flasks.
c) Grow until an OD 600 nm of 0.8 to 1.2. Then induce the culture to express protein by adding 0.3 mM IPTG (isopropylthiogalactoside, MW 238 g/mol) or ~0.1 gram per 1.5 liter flask. This is expensive stuff so use it carefully. Only measure out exactly the amount of IPTG you need for your flasks. Dissolve that amount in about 10 ml of sterile water and divide it equally among your 1.5 L flasks. This stage is called induction. Keep the culture shaking at 37oC.
d) Induction of protein generally takes 3 to 4 hours (but this too depends on your protein). After induction, centrifuge your bacteria in 500 ml bottles in the big Sorvall rotor at 5,000 RPM (bigger than the GSA found on the 2nd floor with Rice, Correl, and Moffat Labs). Do this in batches until all your culture is spun down saving the cell pastes each time. Freeze the cell paste at -80oC.
a) Make Lysis Buffer: 25 mM TRIS-Cl, 2 mM EDTA, pH 7.6. (You may add protease inhibitors like benzamidine or PMSF if you like but add these right before you start to lyse. Make concentrated PMSF solutions in pure ethanol since it is hydrophobic.) Then add lysozyme at 100 mg/ml concentration (or just a tipful from a spatula). Generally the volume lysis buffer is 1/20 to 1/50 the volume of the bacterial culture.Step 5: General protein purification protocol. How to figure out a good scheme? Here is an example of a specific scheme for ubiquitin. A helpful guide from Pharmacia.
b) Resuspend the frozen cell paste as best you can in the Lysis Buffer using a 10 ml pipet or whatever means necessary. Let this suspension incubate for 20 minutes at room temperature, or until the suspension becomes turbid and viscous due to release of the bacteria's genomic DNA.
c) In order to eliminate the extreme turbidity of the suspension, sonicate the suspension to shear the DNA until the turbidity is similar to that of a normal protein solution. Then centrifuge at 18,000 RPM in a Sorvall SS-34 rotor (or ~40,000 x g) for 20 minutes at 4oC.
d) Save the pellet and the supernatent. (If the solution is slighly turbid due to residual DNA, a quick way to shear the DNA is to pass through a syringe with a needle.) Generally your protein is in the supernatent so simply freeze your pellets until you know where your protein is. Protein purification from this supernatent, however, will depend on the properties of the protein: its isoelectric point (pI), size, hydrophobicity, etc. (see below).