Protein Purification and Method Development Using PhyTip Columns
Protein Purification and Method Development Using PhyTip Columns

Optimization and Enhancement of the Purification Process

What is affinity purification?

Affinity resins are designed to be selective to a particular protein tag or protein property or characteristic. Recombinant proteins are normally purified using a tag on the protein and an affinity resin that is selective to the particular tag. HIS tagged proteins are purified on IMAC affinity resins. Antibodies are purified with Protein A or Protein G affinity resins. There are many other types of affinity resins including other tag selective resins or ion exchange resins.
Proteins in a pH environment have a specific charge and can be purified with ion exchange affinity resins. The combination of affinity and specificity has been exploited to generate straightforward affinity purification methods.

What are the basic parameters that can be optimized and enhanced in the PhyTip column process?

Depending on the goals of the researcher, parameters that can be optimized or enhanced include concentration, mass, purity, or activity of the recovered protein, the speed of the purification process and the ability to accommodate various starting sample volumes. The reader is invited to read the Tip Concentrating Effect section for additional details on improving recovered protein concentrations. Some addition information is included in the questions and answers below. Your PhyNexus representative is always for further information. We are happy to help.

Why and how should the capture step be enhanced?

In order to obtain optimum recovery, whether it is concentration or mass amount, it is first necessary to make certain the capture is optimized and as much protein is captured onto the resin bed as possible. Of course this starts with the expression of the protein – it can’t be captured if it isn’t there in the first place. The best way to measure expression of a protein is to run a control protein in parallel with the sample under exactly the same conditions.
Parameters that increase capture include increasing the number of capture cycles and slowing down the capture flow rate. In many cases, adding salt to the sample solution will increase uptake.

Salt reduces the solubility of the protein and can increase the selectivity of the resin for the protein. These salts enhancing protein selectivity for the resin follow the Hofmeister series: increased selectivity effect of anion to decreased selectivity: PO43- > SO42- > COO- > Cl- ; increased selectivity effect of cation to decreased selectivity: NH4+ > K+ > Na+. But many researchers simply use NaCl in the capture with good results. Also, it is important to check the sample pH – uptake of protein is optimized at neutral pH for most affinity resins.

How does washing the resin affect recovery and purity?

When the resin is loaded, both the desired protein drug and impurities are adsorbed on to the column. As the separation method proceeds the buffer strength is increased to wash the resin. Impurities ideally completely desorb from the resin while the material of interest is still completely bound to the resin. Depending on how tightly bound to the resin, the stringency of the wash may be increased to remove as many impurities as possible without removing the desired material and without harming or denaturing the desired material. Pure desired material remains on the column. Finally, at the elution step, the buffer strength is increased so that the desired material completely desorbs from the resin and can be recovered.

In practice, wash conditions, are carefully selected so that desired product remains on the column while undesired materials are removed and washed away. If the target bio molecule to be recovered is a relatively small molecule, the binding is usually relatively high. Tagged recombinant proteins or protein fragments generally have a single point of attachment to the affinity resin. An example is HIS-tagged proteins or fragments which have affinity for IMAC resins. Smaller molecules that have a single point of attachment bind tighter to resin than a larger molecule due to steric hindrance or other factors. On the other hand, if the number of binding sites increases with size, then the selectivity may increase with size, again depending on steric hindrance and other factors. Stringent conditions can be chosen for washing without fear of removing the target bio molecule. With stringent washing, non-specific materials are efficiently removed prior to elution of the pure target material. However, strong elution conditions may be needed for complete recovery.

If the sample bio molecule is relatively large, it may not bind as tightly to the stationary phase. The stringency of the wash is normally reduced to prevent loss of the target material. Conditions for washing are carefully chosen to remove as much non-specific material as possible while still retaining the target material. However, elution is relatively easy and high recoveries from this step are possible, provided sample material was not lost in the wash step. Thus, conditions for capture, wash and elution are developed for each case to optimize the mass recovery, concentration and purity of the bio molecule.

Why and how should the elution step be enhanced?

Removal (elution) of the protein from the resin in the PhyTip column can be surprisingly difficult for some sample proteins. Running a control protein in parallel with the sample under exactly the same elution conditions is an excellent measure of elution efficiency, provided the control protein is similar in structure to the sample protein. Large bulky proteins tend to elute easily from the column whereas smaller proteins are more difficult.

Standard elution volumes are 3x of the resin bed volume. A higher concentration of protein can be achieved by reducing the elution volume to 2 x of the resin bed volume. Many researchers will perform a second elution and combine the fractions. This will increase the mass recovery but will reduce the overall concentration of the recovered protein.

How can the highest concentration of recovered protein be achieved with all protein in the sample recovered?

First, use a column with sufficient capacity to capture the entire amount of protein. Passing the sample over the resin bed several times allows as much capture of the protein from the sample as possible. Using a slower capture flow rate also increases the residence time of the sample in the resin bed which increases the capture of the sample. In cases where the sample volume is significantly larger than the total volume of the PhyTip tip, it is better to use more capture cycles rather than a slower flow rate so that eventually all of the sample protein passes though the resin bed and is captured. Better still the sample volume may be split into several volumes
and each sample volume captured in succession.

In some cases the type of resin can be important. For example, Pro A resin has a higher capacity than Pro G resin for many types of antibodies. Also, use sufficient sample target protein so that all of the resins functional groups have captured protein attached. If necessary, follow all of the optimization procedures for number of capture cycles and flow rate. Finally, using a smaller elution volume increases the concentration of the eluted protein.

How can a normalized concentration of recovered proteins be achieved?

Recovering a normalized concentration of protein (the concentration of protein recovered from the tip is uniform regardless of the starting sample concentration) can be useful in many cases.
Many assays, especially cell based assays, require a minimum and uniform amount of purified protein. This can be difficult to achieve if the expression efficiency or effectiveness of the protein is unknown or known to vary for a series of proteins. PhyTip tips are unique in their ability to normalize concentration of purified protein to a predictable level. Starting sample protein concentrations that vary widely can still be brought to a uniform concentration in the purified form. The resin bed is overloaded even with the lowest expressed protein by using sufficient sample concentration and volume. The first step is to use the smallest possible bed volume that is appropriate for the assay. In most cases, this will be the 5 μL bed volume. Overload the resin bed by a factor of two or more. If the concentration of a sample or series of samples is unknown, then it is best to process enough sample volume so that the lowest expected expressed sample has sufficient volume to overload the column and then treat all samples in the same way. It may be necessary to process several aliquots of the sample in series to overload the column.

How are bio molecules taken up and eluted from affinity columns and other columns?

The interaction of bio molecules with most chromatographic phases can be characterized by sharp isotherm “on-off” chemistry. Under any set of buffer conditions the bio molecule is either adsorbed to the chromatography material or desorbed from the material. This appears to be true for ion exchange, ion-pairing, affinity, hydrophobic interaction, reverse phase, hydrophilic interaction and normal phase material. However, this controlled interaction can only be used when the interaction of the bio molecule and column come to equilibrium under the specific buffer conditions.

What parameters are important for purified protein suitable for structural analysis?

Several parameters are important. High concentration for structural analysis is important of course, but the recovered protein must also be in the suitable buffer environment for forming crystals and have the correct folding and protein activity. Therefore it is necessary to follow the guidance above, but also to perform the operations in parallel to screen different buffers.

A two-step process for achieving ultra-high protein concentration is possible with the PhyTip tip but is beyond the present discussion. Contact Phynexus customer support for more details.

How can the highest mass amount of recovered protein be achieved?

Use the largest resin bed volume so that the resin bed is not overloaded (there is no protein in the capture flow through). The capacity of the resin bed is proportional to the bed volume. If the sample volume is large, be sure to process (with sufficient cycling) an aliquot of sample completely with each capture cycle and then repeating the cycling process with another aliquot until the entire sample is processed. Another strategy is to process only part of the total volume with each capture cycle but use several capture cycles. Eventually the sample protein will travel through the bed and be captured. Typically 10 or more capture cycles is used in this strategy. If the resin bed capacity is severely overloaded, two PhyTip columns may be used. Generally two elution aliquots should be used to recover the entire amount of protein from the resin bed.

How can the highest purity of recovered protein be achieved?

Use progressively higher concentrations of the wash solvent to remove nonspecific bound protein and check the purity of recovered protein using gel electrophoresis. The wash can remove the captured protein as well as nonspecific bound proteins and other materials. This is especially important in PhyTip column extractions because the ratio of wash solvent volume to bed volume is extremely large. So this process should be done carefully using only lowest concentration of solvent necessary for cleaning. At least two aliquots of wash solvent are necessary. More aliquots may be used but usually are not necessary.

How can the highest activity of recovery protein be achieved?

It is important that activity of a protein is reduced by denaturing or other means. A unique aspect of PhyTip columns the absence of high surface area frits that can harm proteins. But it is also important to use buffer conditions that are not denaturing in any of the processes including washing and elution. Generally this means that the buffer pH is kept neutral and at high ionic strength. Many researchers purify in parallel and screen expression conditions and purification conditions. Activity can be tested with enzymatic assays, ELISA, SPR or a functional test.

How can the purification process be shortened?

One answer is to process samples in parallel as much as possible. Of course reducing the number of cycles will reduce time. Often just 2 or 3 cycles are sufficient for the capture process. But use fewer capture cycles or use a fast flow rate but only if necessary. Usually a larger bed volume column will capture the limited sample in a shorter time period. If possible reduce the wash and elution/enrich cycles before the number of capture cycles is reduced. Often, for washing, usually only 1 cycle of each solvent is adequate. Lowering the number of capture cycles down to 2 cycles is acceptable in almost all cases but in some cases this could lower the amount of protein captured and increase the deviation of the recovery from sample to sample.

How can the smallest sample volumes be processed?

The ability to process small sample volumes enables the possibility of the expression of small volume of proteins, reducing the amount of material required to produce the desired protein.
Normal sample volumes range from 0.2 mL to 1 mL, but samples can be as large as several milliliters or as small as a few microliters. Because of the low dead volume and low resin bed volume of the PhyTip tips, very small elution volumes can be used to elute protein from the resin bed. Very high protein concentrations are achieved with these extremely small elution volumes. The concentration of the protein increases by using decreasing elution volumes (although less mass amount of the protein may be recovered). Use the smallest resin bed volume possible and use the smallest elution volumes (the concentration increases with decreasing elution volume).

How should large sample volumes be processed?

Two different methods are normally employed. Sample volumes larger than the tip chamber volume can be processed simply by inserting the column in the sample and processing with sufficient number of cycles so that any sample protein in the sample has a chance to contact the resin bed. A general rule is to double the number of capture cycles exponentially for every chamber volume of sample. Thus if a 1 mL sample in the 1000+ column (with a 1 mL sample chamber) uses 4 capture cycles, then a 2 mL sample using the same column should use 8 capture cycles, a 3 mL sample should use 16 capture cycles and so on.

A better method is to split the sample into as many aliquots as necessary and process each aliquot in succession until the entire sample is captured. Then proceed on as normal with the wash/purification step and the elution/enrichment step.

What factors are important in PhyTip sample capture?

Of course the affinity chemistry must be suitable to selectively capture the protein of interest.
For example, Protein A resin will capture Mouse IgG2a but will not capture Mouse IgG1. Refer to the Protein A and Protein G affinity information and product insert sheets to identify the correct affinity resin for a particular type of antibody capture. ProPlus and ProPlus resins have selectivity that combine Protein A and Protein G. IMAC affinity resin is used to capture HIS tagged recombinant proteins. The sample pH must be correct – in most cases, a sample of pH 7 is desirable for capture. For some sample proteins, it is important to increase the column bed residence time to compensate for low-affinity interactions. Capture residence time is increased by decreasing capture flow rate and/or increasing the number of capture cycles.

What is the maximum loading capacity for a PhyTip Tip? For example, what is the loading of a Protein A PhyTip column?

When using the largest bed volume 1000+ PhyTip tips, up to 2 mg of human IgG can bind to the
Protein A affinity resin provided the sample contains enough protein and proper loading procedures have been followed. To fully load the resin bed, at least a slight excess protein is needed in the sample. High loading is achieved with slower flow rates or more loading cycles; however, if the sample protein excess is large, flow rate and number of loading cycles are less important. Contact your PhyNexus representative for further information.

What size proteins can I purify with the PhyTip system? For example, can I purify protein complexes that are very large?

Most proteins that researchers study are in the 5 to 200 kDa size range. Interestingly, as the protein size increases, the larger, more bulky proteins may not stick as tightly to the affinity resin and the conditions for purification are usually less stringent to prevent loss of protein.
Nevertheless, these proteins are purified quite easily. Protein complexes however are much larger e.g. 1 MDa to perhaps the 8 MDa range. Proteins in a complex held in a strong core of proteins (proteins are tightly associated with each other) are usually captured by packed bed columns and will also be captured by the PhyTip columns. Transient (non-core) proteins are often difficult to isolate in packed bed systems; they are often too large and fragile to survive the process of flowing through the packed bed. The gentle action of the PhyTip system will enable purification of transient proteins in protein complexes. Each system should be examined on a case by case basis.

What are affinity tags?

Recombinant proteins are generated by introducing an extrachromosomal DNA vector (plasmids containing the gene of interest) into cells. Then the plasmid vector utilizes the cell’s machinery to express the gene to produce the protein of interest. Affinity tags attached to the
N-or C-terminus of the protein are expressed along with the recombinant protein. These affinity tags make it possible to perform affinity purification with the appropriate affinity resin. After capture and column washing, the recombinant protein is recovered by eluting the column with a buffer solution. In some cases, tags can be enzymatically cleaved of the column. An example of this is engineering TEV sites into the DNA vector in addition to DNA expressing the tag. The affinity resin will capture the protein with the tag while leaving the TEV site available for cleaving. Some tags can be attached to proteins directly (without plasmid vectors). An example of this is the chemical attachment of the biotin tag to lysine residues on the sample protein.

What are some common affinity tags and their counter ligands?

6-Histidine peptide residue (6-His) tag will bind to IMAC affinity resin containing divalent metals including Ni(II) or Co(II). Glutathione S transferase (GST) tag will bind to glutathione (GST) affinity resin. Biotin tag will bind to Streptavidin affinity resin. FLAG tag will bind to anti-FLAG antibody affinity resin. Maltose Binding Protein (MBP) tag will bind to amylose affinity resin.
Myc tag binds to Anti-Myc antibody affinity resin. Calmodulin Binding Protein (CBP) tag binds to Anti-calmodulin binding protein antibody affinity resin. HA peptide tag binds to Anti-HA antibody affinity resin.

What are the major challenges to affinity purification?

Affinity purification methods often struggle to maintain or increase the concentration of the protein being purified while at the same time providing a protein that is pure and active. This is especially true as the amount or volume of protein being purified is decreased. It can be very difficult manipulating microliter volumes of sample. Finally, it is difficult to routinely purify large numbers of samples in parallel in a laboratory environment. PhyTip column technology is designed to easily overcome all these challenges.