The polyhistidine tail on the protein of interest allows it to be purified from other proteins using metal affinity resin. Using the proper amount of resin is important: too much resin may indiscriminately bind to proteins that lack the polyhistidine tail, resulting in impure protein, but too little resin will cause some of the desired protein to be lost as the resin is overloaded, resulting in reduced yield.
The elution buffer used to elute the polyhistidine-tagged protein during the purification procedure (containing sodium phosphate, sodium chloride, and imidazole) is likely not an optimal buffer for your assays. Therefore, the protein must be removed from elution buffer and placed into a storage or assay buffer. The method of purification used to desalt protein samples—gel filtration chromatography—is conceptually important to understand. The principles of it are described in Chapter 5 of Mathews, Van Holde, Appling, and Athony-Cahill or Chapter 6 of Voet and Voet.
Deep Thoughts on Buffers
You’ve spent a lot of time learning about buffers and pH in classes since high school. But do you remember what a buffer is and what the purpose of a buffer is? If you don’t, you’ll want to brush up on this before continuing.
You’ve used and made a variety of buffers already but what makes a good buffer? For example, buffers for SDS-PAGE and polyhistidine affinity purification have prescribed recipes optimized for each specific purpose. Now, it’s up to you to select and make your own buffers for your experiments. At the very least, you’ll need to select a buffer suited for storing your protein in which to desalt. What makes a good buffer in this case? It depends on what qualities you want in your buffer. In this case, you probably want a buffer in which your protein in stable. In order to avoid changing buffers again, you also likely want a buffer that will not interfere with your assay of choice. There may be additional qualities to consider when choosing and making your buffer.
Deep Thoughts on Protein Purification
There are many different ways in which proteins can be purified. In this course, we are using one of the simpler and more common ways to purify recombinantly expressed protein- polyhistidine affinity purification. How does this method actually work? What does it rely on in order to work effectively? If you are having problems with your purification, what could be going wrong and how might you troubleshoot?
There are many other purification techniques you could use other than polyhistidine affinity purification which rely on size, charge, or the affinity of other molecules as an alternative purification technique or in addition to polyhistidine affinity purification in order to obtain a purer sample. What are some of these techniques? What would they require in order to effectively purify your protein of interest and what would be the pros and cons of using them to purify your protein of interest?
- Microfluidizer (available for BB494)
- Sonicator with a power source and ear protection (alternate method of lysing)
- Oak Ridge high speed centrifuge tubes
- Sorvall and SS34 rotor
- Poly-prep columns –Bio-Rad (reusable)
- Prepacked PD-10 desalting columns from GE Health Care-reuse these
Protein Purification and Desalting
- BD Talon Metal Affinity Resin (teams will receive aliquot of resin in long-term storage buffer. Resin will need to be equilibrated).
- 5X Talon Equilibration Buffer (250 mM sodium phosphate, 1.5 M NaCl pH 7.0). Important Note: For using the microfluidizer, prepare a 0.5 L stock of 5X. Dilute to 1X as needed each time you purify.
- 1X Talon Equilibration/Wash buffer pH 7.0 (50 mM sodium phosphate, 300 mM NaCl) (also referred to Tractor buffer in Talon manual)
- Talon Elution buffer, pH 7.0 (50 mM sodium phosphate, 300 mM NaCl, 250 mM imidazole) (make 100 mL or less)
- Hen egg white T4 lysozyme if sonicating
- Cell pellets from spun down expressions
- Bradford Protein Assay Reagent and bovine serum albumin protein standard
Suggested Resources and Protocols
- Protein purification buffer preparation: The manual for TALON Metal Affinity Resins is posted on Canvas site. Note: Talon manual refers throughout to Tractor Buffer—you will make your own Talon resin Equilibration/Wash and Elution buffers (see list above).
- Protein Desalting: The manual for using PD-10 Desalting columns can be downloaded from the manufacturer’s website and is also posted on Canvas.
http://www6.gelifesciences.com. (inactive link as of 5/19/2021) Select a buffer for desalting that is compatible with your assays. Make sure you understand the general concept and use of terms equilibration and elution buffer.
Deep Thoughts on Protein Stability
Protein stability is applicable to this course in multiple ways. First, you’ll want to consider the stability of your protein over time in the context of conducting your experiments. Second, stability (over time or perhaps thermostability) may be a feature of your enzyme that you’ll want to assess as part of your research. Changes in stability are often of interest and increased thermostability is often a desired outcome when engineering an enzyme.
First, we will focus on stability in terms of maintaining the integrity of your protein. You will be expressing and purifying your native protein as early as Week 3 of the term. Do you expect this purified protein to have the same activity and behave the same once you get to your final assays in Week 9? What can you do to maintain the integrity of your protein over time?
One key factor to consider is how your protein is stored; understanding and choosing conditions under which your protein may be stored without appreciable loss of activity is essential for effectively evaluating your protein. In order to maintain maximum activity and obtain consistent results from lab period to lab period, how should you store your protein? At what temperature? In what buffer? At what concentration? How should your protein be handled when you are using it?
If you are observing loss of activity over time, what could be happening to your protein to reduce its activity? What could you do to mitigate the loss of activity? What could you do to limit the effect of the loss of activity of your protein over time on your results? Aside from measuring reduced activity, how might you detect that the integrity of your protein is being reduced with time?
Additionally, if you want to assess the stability of your protein as an additional feature, how might you go about doing so? What is an appropriate assay and how might you challenge your enzyme?