Protein purification is a challenging endeavor for many scientists and it usually takes time and sweat to get to your end results. Calling a protein purification a success can mean something different for everyone. Maybe it’s about getting a single band on an SDS-PAGE? It could be isolating a protein that is free of contaminants and has retained its activity. Sometimes, it’s about isolating as much material as you possibly can as you have a ton of experiments that need to be run with that protein. For some, completing the last step of a long drawn out protocol whatever the outcome may be enough to cry out success!
With any experiment, it’s important to standardize the steps to ensure consistent results. Let’s take a look at three metrics that are commonly monitored and should be measured during any protein purification.
An old saying in biochemistry is “never waste pure thoughts on impure proteins.” Indeed, in some research and therapeutic applications, even minor impurities can cause major problems. Defining the levels of purity of your protein sample should be dependent upon the impact it has on downstream tests that are performed. It’s pretty safe to say that striving for the highest purity is likely going to make your next steps go much more smoothly.
Often, researchers will combine different protein purification methods that apply unique separation principles such as size or charge of the proteins being separated. A little bit of forethought on what you plan to do with your purified protein will dictate the strategy for you to use.
The amount of protein you recover is also a factor when it comes to measuring the success of your protein purification. Researchers want to make sure they can retain as much of their target protein as possible. However, each step in a protein purification protocol usually results in some degree of product loss. So, if a high yield is what you’re aiming for, try to limit the number of steps during your protein purification. Knowing certain physical or chemical properties, such as molecular weight, isoelectric point, and optimal pH, of your target protein can be very helpful when determining the necessary steps.
Protein loss may also occur prior to chromatography, as the expression of a protein might be suboptimal. In this case, you can try several approaches in parallel that can speed up the process and increase the chances of success, such as:
- Testing several expression systems (vectors, cell types, and/or strains)
- Testing different induction conditions (OD, temperature, oxygen, and/or inductor concentration)
- Checking the efficacy of sonication or other means for cell disruption
- Checking codon usage of your expression system
Stability and activity
Lastly, the stability of a protein in terms of activity and aggregation is an important criteria for success. If you’re planning to perform functional studies with your purified protein product, it’s an absolute requirement that it retains its biological activity and structural integrity after purification. With these requirements in mind, it’s important to:
- Transfer the cell extract as quickly as possible to a stabilizing environment, away from any proteases released during the cell disruption step.
- Determine the stability window in terms of pH, ionic strength, oxidation-reduction reactions, temperature, and presence of additives, before purification.
- Evaluate stability during and after the purification process. This may require running a gel or monitoring the presence of your protein.
- Minimize the purification process and storage time.
So take the time to think about what defines success for your project. And keep in mind that, “Failure is success if we learn from it” — Malcolm Forbes.