Every protein is unique and thus every protein has its own set of production and purification challenges – many of which cannot be predicted. Therefore to successfully produce and purify your favorite protein you need to know and understand these five unpredictable protocol variables (X factors). Tweaking these X factors just might be the difference between a miserably low yield and success.
X-Factor #1: Your Expression Construct
The main goal of a good expression construct is to express your protein so it folds into an experimentally useful conformation. This is the most important factor for successfully producing and purifying your protein! And it requires not only intelligent design but a bit of dumb luck too.
Factors to consider when designing and testing your expression construct:
- You must clone your protein into a bacterial expression construct! Not a mammalian expression construct, as the promoters are completely different.
- After choosing your vector, make sure you understand the promoter system, the chemical to use for induction, and that you are using the correct strain of bacterial for that induction system.
- Take into account your protein’s function and binding interfaces, especially focusing on the binding interfaces near your tag or antibody epitopes.
- If you also use your expression construct to add tags to your protein, make sure they are placed where they won’t interfere with biological function. To successfully place tags requires an acute understanding of the biological function of your protein and trial-and-error testing.
- Decide what you will do with any added tag. Will you leave them on or remove them before using your protein?
- Check that the linkers used between the protein and tags are made of hydrophilic sequences – this will help with solubility and folding.
X-Factor #2: Your Choice of Bacteria
The health of your bacteria can make a big difference! To ensure optimal health of your bacteria and their ability to make protein, use fresh cultures and watch your OD carefully and induce only during log-phase growth. If you don’t start with healthy bacteria and prevent overgrowth by watching the OD, not only can you interfere with good protein production, but you can also cause outgrowth of bacteria that don’t contain your plasmid.
The type of bacteria is also very important. While the E. coli line BL21 is the old standby, there are many commercially available bacteria designed to enhance yield under various conditions – so shop around.
There are commercially available strains engineered to:
- Increase mRNA stability.
- Reduce protein degradation.
- Prevent uninduced expression of your protein. Many proteins can be toxic and these bacteria have a more tightly regulated induction system to prevent death of the cells containing your vector.
- Overcome less-than-optimal codon usage. These bugs are more forgiving if your clone’s codons are optimized for mammalian expression than bacterial.
- Aid proper protein folding by
- containing protein systems that enhance disulfide bonding or
- containing more chaperones that help fold your protein into something soluble and stable or
- containing “tunable promoters” that regulate the amount of protein to be folded at one time – especially good for hard-to-fold proteins such as membrane proteins.
X-Factor #3: Protein Expression Conditions
An optimal expression construct and bacteria strain do not guarantee success. You must also have optimal expression conditions! After all, the biggest barrier to making any protein is to get it to express, preferably in a folded state (instead of rapidly degrading or pooling in completely insoluble clusters).
Sometimes, to achieve good folding or protein production, all you need to do is to control the folding rate. After all, the slower the bacteria produce protein, the less folding/metabolic stress your bacterial systems experience and the happier your bacteria and their yield. To slow down the rate of protein production, you reduce the temperature that your bacteria are grown in after protein induction. For example: Instead of inducing at 37°C for several hours, do 20°C overnight. I’ve even see others do 4°C (yes a flask stirring in the cold room) for weeks! Optimizing the rate using altered temperatures is the first thing you should experiment with to increase your protein induction yield.
X-Factor #4: Protein Extraction and Solubilization
Your protein must be soluble in order to purify it. And no matter how easy your protein is to make, some of it will be insoluble, or if you are really unlucky perhaps all of it. To solubilize protein, typically we begin with detergents before moving on to denaturing urea – this is to preserve as much of your protein folding as possible. For detergents, Triton is a good first choice, as it is somewhat mild. But if Triton doesn’t work I recommend going straight to the detergent sarkosyl: it just work like magic. And if sarkosyl can’t significantly solubilize your protein then move on up to urea – after all it is better to get poorly-folded something than nothing at all.
X-Factor #5: Protein Cleanup, Concentration and Storage
Few things are worse than making a lot of quality protein, going home feeling all proud and accomplished, only to return the next day to find it ‘crashed out’ of solution and your sample looks like a mini snow globe. Crashing out (precipitating) can happen during a number of steps: elution, dialysis, concentrating, storage or while defrosting previously frozen aliquots. So watch your protein solution carefully!
If your protein DOES crash out of solution you may need to change your dialysis solution or add glycerol to stabilize it. Also consider storing your protein solution at 4°C instead of in the -80°C freezer, or vice versa. Worst case scenario: You will need to make your protein solution fresh every time you need it (no storage!).
But before you chuck your precipitated protein out. Know that even if your protein crashed out of solution, it still may be at a usable concentration (spin out the precipitant and quantitate to check). Alternatively, if you are making an antibody, a denatured peptide might be more antigenic.
While successful protein production and purification often seems like luck, with my five unpredictable variables (X factors) I am sure you can systematically create your lucky protocol