Well, most of us have been through this situation wherein we end up with a lousy protein prep. It adds to our misery if it's already a standardized protocol and yet we don't seem to get it right. If that's what's been happening to you, then you are in the right place.

I have been working with proteins for the last seven years and have grown to understand and know them closely. Purifying proteins has been an indispensable part of my daily routine. While purifying is just one aspect, getting them at required concentrations and ensuring their active state is a tremendous challenge. In today's post, I am going to let in a few tricks that will help you deal with exactly that! I am going to let you guys in into a little secret!

When you work with proteins it's important to realize that proteins are exactly like humans. Each protein has a distinct personality. Some are moody, some are stubborn while some are easy going and friendly. Some proteins like to form groups, we call them oligomers while some work in isolation. Some love to collaborate while some are happy singletons. It's up to us to figure out what our little man is all about! Can we paint the most accurate picture of the protein in question?

Before we start purifying our protein (assuming its a recombinant protein from a bacterial expression system) there are many basic parameters that we often tend to neglect. Few of the most overlooked but important parameters is the concentration of the inducer, time and temperature after induction and the O.D. (optical density of the bacterial cells) during induction. Theoretically, an O.D. of around 0.6 to 0.8 is considered accurate for induction. Although while the accurate is an average prediction one can in principle change this with the nature of the recombinant protein you are expressing. Proteins can be allowed to induce at lower temperatures for longer durations or at higher temperatures for shorter periods of time. For instance, bigger proteins might need more time for folding and hence can be induced at lower temperatures for longer durations. This can also be employed for expressing oligomeric or multi-protein complexes. On the other hand, cytotoxic proteins can be induced at higher temperatures for a short duration before lysing the cells. This is a key trick to avoid proteins from going into membrane fractions or extremely low expression levels. If you are sure that the protein is quite happy and well-folded but still shows fewer expression levels, the concentration of the inducer might be a crucial parameter you might want to tweak. Changes required there would be highly dependent on the expression system one uses and can be easily optimized.

Once the protein expression and folding possibilities have been explored and optimized enough, the very next important parameter is to choose an appropriate buffer system. Most of us tend to stick to the traditionally used favorites- Tris-HCl, HEPES, Citrate and the Phosphate buffer systems. Based on the overall charge of the protein, in particular, the pI of the protein and the method of purification selecting a suitable buffer plays a crucial role in the overall process. Proteins with acidic pI favor buffers that maintain pH in the basic range like the phosphate and Tris-HCl while proteins with basic pI do enjoy citrate, HEPES, and acetate buffer systems. Sometimes, it is wiser to use buffers like Tris-HCl (overall positive charge) for proteins with very low acidic pI and not phosphate. The solubility of a protein in a particular buffer system can be verified at smaller scales to avoid hassles in the large-scale purification process. I am pretty sure that you already do all of this. But do not forget the following key points while checking the pI of your protein-

(i) Check the pI for the entire length of the protein. Do include the residues from the tag if present (For example, -His tag, FLAG tag etc).

(ii) Always include the residues contributed by the expression vector as well if they are a part of the reading frame (the protein can be tagless although sometimes residues contributed by the expression vector should not be overlooked- they contribute to the overall pI of your protein).

(iii) For protein complexes, include the sequence of all the proteins forming the complex to calculate the overall pI.

Tips:

(1) Adjust the pH of the buffer after the addition of salt since salts are known to alter pH (pKa changes from 8.06 at 25°C to 8.85 at 0°C).

(2) HEPES interferes with the Lowry protein assay (not the Bradford assay). Since HEPES can form radicals under a range of conditions its use should be avoided in systems where radicals are being studied.

(3) Phosphates tend to crystallize at lower temperatures and higher strengths. Also, phosphate buffers are not compatible with divalent cations like Mg²⁺ ions.

(4) Certain buffers like Tris have reactive amines and can interfere in enzymatic reactions.

The method of purification also plays an important role in buffer selection. In particular ion-exchange chromatography demands special attention to buffers than ordinary. Selecting the correct ion-exchange resin and then accordingly deciding the buffer plays a significant role in getting a successful prep done. You can find more on this in my post on Ion-exchange chromatography for protein purification.

Once the central buffer system is decided, it's crucial to set up the appropriate ionic strength. No one likes their buffer too salty or way too bland. More on this soon...

"Yikes!! It’s too salty!!! ".....Now you don't want your protein to say that do you?

Coming to how we decide the accurate ionic strength of the buffer while we purify our protein is an easy yet extremely arduous task. There are majorly two parameters that one needs to consider;

(1) The ionic strength

(2) The correct ion for your protein

Ionic strength is a very important parameter since it is responsible for improving protein folding, solubility and protein-protein interactions. Generally a broad range screening of salt molarity is sufficient to decide the ionic strength of the buffer required for protein purification. Ionic strength can be determined using the formula,

Ionic strength of aqueous salt solution is expressed as:

μ=0.5*(Σv²C)

where v is the valency of each ion and C its concentration.

For instance, for 1 M NaCl, μ=1.0 (van Oss, 2008)

Identifying the ion of choice for you protein necessitates screening of the protein in different buffers with varying ions. For instance you might not want to stick with NaCl (Sodium chloride) always but try out other salts like KCl (Potassium chloride), MgCl₂ (Magnesium chloride), ZnCl₂ (Zinc chloride), MnCl₂ (Manganese chloride) etc. There have been instances where the activity of an enzyme has showed improved results in KCl or in the combination of KCl and MgCl2. Once you have screened as to which ion/s suits your protein best you can be rest assured that your protein is going to be stable while purification.

Tip:

Always adjust the pH of your buffer at the temperature of protein purification. Ceratin buffers like the Tris buffer are known to have a very high dpKa/dt. In other words, these buffer systems show a change in pH with temperature.

Once we have our buffer and salt in place, its time to focus on to additives. Additives are several other components apart from the main buffering agent that can play a role in protein quality. Addition of compounds like protease inhibitors, reducing agents, osmolytes, α-helix stabilizers and charged amino-acids can dramatically improve protein solubility.

Which additive to use is dependent entirely on the protein we intend to purify. For instance, the protein composition might provide us with clues whether you would need a reducing agent at all. A Cysteine-rich protein is likely to have reduced or oxidised forms of Cysteine and a reducing environment is more suitable for the protein’s stability. If one is experiencing loss of concentration due to degradation by cellular proteases, protease inhibiters are highly valuable. Higher concentrations of proteins can be achieved via addition of osmolytes and charged amino acids. Protein folding, an important aspect of protein stability and solubility can be aided by the use of certain molecular chaperones.

Tip:

Do NOT use additives if not required, since they can be a probable hurdle for dowstream processes.

Always remember that the best purification buffer is an accurate mix of the buffering agent, salts and additives. A bit of experimenting and testing is all that is required to paint a perfect picture of our protein of interest. With all the luck, stay busy experimenting!!

Proteins in the physiological context are seldom made of single polypeptide chains but more often than not are comprised of multiple subunits in the form of complexes. To characterize such complexes both structurally and functionally it is crucial to purify them in the desired stoichiometry, a feat easier said than achieved. If that's what's been bothering you for a while now, don’t worry, I am going to let in a few tricks that will help you deal with exactly that!

Traditional approaches and their shortcomings

Traditionally, protein complexes are obtained through Affinity chromatography or in vitro assembly of functional subunits. In Affinity chromatography, one of the subunits in the complex is suitably tagged (Histidine tag/Strep tag etc.) and co-expressed with the other subunits. The lysate is then incubated with the immobilized resin that acts as an affinity support. The complex binds specifically to the resin through the tagged subunit. After a brief washing step the complex is eluted suitably (elution protocol depends on the nature of the tag) (Figure 1A). Alternatively, multiple subunits are purified separately and then incubated for complex formation in vitro, provided one can purify all the subunits independent of each other (Figure 1B). Although these methods work fairly well, in both the cases, the assembled complex needs separation from the non-assembled or partially assembled subunits – which is a grim task! But do not worry, by the time you reach the end of this article, you will have a way out for this!

The twin-tag system

One of the most effective strategy of purifying protein complexes with 1:1 stoichiometry is the Twin-tag system. For a clearer understanding of this method, let’s consider a protein complex made of two subunits, A and B (Figure 2). Both A and B are tagged with distinct affinity tags and co-expressed in the host cell. They are then purified as a complex AB by consecutive two-step affinity purification protocol. In the first affinity step, the complex is bound to resin 1 via the affinity tag on A. After washing, the bound complex is eluted from the resin. The first eluate thus consists of free A and the AB complex that was bound to the resin. This step gets rid of free B since the tag on B does not bind resin 1. Subsequently the eluate is incubated with resin 2 (specifically binds to the tag on B). The binary complex AB now binds to resin 2 via the tag on B and free A is washed off in the washing step. The complex then is eluted off the resin using a suitable elution protocol. This system thus gets rid of both, non-assembled and partially assembled complexes. If your protein complex is made up of multiple subunits, you don’t have to worry. Just use different affinity tags (enlisted here) and appropriate number of affinity purification steps in your protocol.

Getting rid of the tags

If required, the tags attached to the subunits can be removed using highly specific proteases. In that case, just ensure that you have introduced a protease cleavage site in between your tag and the protein. This is not so a difficult task, since most plasmids used for cloning contain cleavage sites for specific proteases that are commercially available. Saying that, it is important to emphasize that use proteases with higher efficiency and mutually exclusive specificities to avoid them from chewing up through different protein subunits.

Proteolytic cleavage of the tags can be performed during purification (on-column) or later. On-column cleavage of tags saves time and is a good alternative for eluting the bound complex from the resin. But the method I personally prefer is to remove the tags after the purification step. Although this method increases the purification time, it ensures efficient cleavage and monitoring the cleavage pattern is easy. If you are cleaving the tag for the first time, I highly recommend you to do so after purification. It will help in the optimization of the protease activity by tweaking parameters like the time required for cleavage and buffer conditions. These proteases can then be separated by passing the sample through a gel filtration column or other suitable methods. For more information on the twin-tag system and different proteases please check here. Until then, enjoy purifying your complex with the perfect stoichiometry!

References:

1. Frey S and Göhrlich D (2014) Purification of protein complexes of defined subunit stoichiometry using a set of orthogonal, tag-cleaving proteases. ‎J. Chromatogr. A 1337:106-115.

2. Kimple M E et al (2015) Overview of Affinity Tags for Protein Purification. Curr Protoc Protein Sci 73: Unit-9.9.