BCH5425 Molecular Biology and Biotechnology
Spring 1998
Dr. Michael Blaber
blaber@sb.fsu.edu 

Protein Purification: Gel Filtration, Affinity and Hydrophobic resins; Preparation of Resin, Plumbing

Gel filtration

Gel filtration does not rely on any chemical interaction with the protein, rather it is based on a physical property of the protein - that being the effective molecular radius (which relates to mass for most typical globular proteins).

• Gel filtration resin can be thought of as beads which contain pores of a defined size range.
• Large proteins which cannot enter these pores pass around the outside of the beads.
• Smaller proteins which can enter the pores of the beads have a longer, tortuous path before they exit the bead.
• Thus, a sample of proteins passing through a gel filtration column will separate based on molecular size: the big ones will elute first and the smallest ones will elute last (and "middle" sized proteins will elute in the middle).

• If your protein is unusually "small" or "large" in comparison to contaminating proteins then gel filtration may work quite well.

Where will a protein elute in a gel filtration experiment?

• There are two extremes in the separation profile of a gel filtration column.
• There is a critical molecular mass (large mass) which will be completely excluded from the gel filtration beads. All solutes in the sample which are equal to, or larger, than this critical size will behave identically: they will all eluted in the excluded volume of the column
• There is a critical molecular mass (small mass) which will be completely included within the pores of the gel filtration beads. All solutes in the sample which are equal to, or smaller, than this critical size will behave identically: they will all eluted in the included volume of the column
• Solutes between these two ranges of molecular mass will elute between the excluded and included volumes

• In gel filtration the resolution is a function of column length (the longer the better)
• However, one drawback is related to the maximum sample volume which can be loaded. The larger the volume of sample loaded, the more the overlap between separated peaks. Generally speaking, the sample size one can load is limited to about 3-5% of the total column volume.
• Thus, gel filtration is best saved for the end stages of a purification ,when the sample can be readily concentrated to a small volume.
• Gel filtration can also be used to remove salts from the sample, due to its ability to separate "small" from "large" components.
• Finally, gel filtration can be among the most "gentle" purification methods due to the lack of chemical interaction with the resin.

Affinity chromatography

Affinity chromatography is a general term which applies to a wide range of chromatographic media. It can be basically thought of as some inert resin to which has been attached some compound which has a specific affinity for your protein of interest.

• Thus, a specific antibody attached to an inert resin would be a type of affinity chromatography.
• Other examples might include: a protease inhibitor attached to some matrix, designed to bind a specific protease
• a cofactor bound to some matrix, designed to bind to a particular enzyme
• a metal ion bound to a matrix, designed to chelate a protein with a metal binding site, and so on.

In each case, the type of resins used and the method of attachment may vary, as will the method of elution. One generalization regarding method of elution is that the bound ligand can be competed off of the column's functional group by including in the elution buffer a high concentration of the free functional group. For example, if the functional group of the column is a cofactor, then the bound protein can be competed off the column by passing a buffer containing a high concentration of cofactor (or cofactor analog) through the column.

Other methods of elution include changing the buffer conditions such that the protein is no longer in the native state (since it is the native state which confers the structure required for the specific binding interaction). This can be achieved by changing pH or by adding denaturing agents such as urea or guanidine.

With affinity chromatography, typically the purification achieved in a single step can be dramatic - on the order of several thousand fold. Single step purifications with specific affinity columns are not unheard - in fact it is an ideal goal of purification - a matrix which recognizes only the protein of interest and none other.

Hydrophobic resins contain a non-polar functional group, such as an alkane or aromatic group.

• Many proteins are able to sequester such groups on their surface and this exclusion from solvent provides the basis of the binding energy (i.e. the "hydrophobic effect").
• This interaction is enhanced by increasing ionic strength, such that proteins may bind under high salt conditions and elute under low salt conditions.
• As such these columns may be used to not only provide purification, but to desalt samples (for example after an initial ammonium sulfate precipitation).
• It is usually not possible to predict in advance which particular resin will bind a given protein, this is usually determined empirically. However, the longer the alkane, or the larger the aromatic compound, the stronger the binding typically will be.

 Due to the nature of hydrophobic interactions and ionic strength, hydrophobic chromatography and ion exchange chromatography can be conveniently used sequentially. For example, after ion exchange the protein is in high salt conditions, thus it can be loaded directly onto a hydrophobic column. Conversely, a hydrophobic column is eluted in low salt, which is a requirement for binding to an ion exchange resin.

A distinction should be noted between hydrophobic interaction chromatorgraphy and reverse phase chromatography

• Hydrophobic interaction chromatography is performed in aqueous solvent conditions and changes in ionic strength are used to elute the column. The protein typically binds in the native state via hydrophobic groups located on the surface of the protein. The native state is retained during the elution conditions
• Reverse phase chromatography utilizes a hydrophobic solvent (typically acetonitrile) and the binding of a ligand is a function of the phase partition between the hydrophobic nature of the solvent and column functional group. Proteins are typically denatured in such solvents and bind due to the hydrophobic nature of the entire polypeptide sequence. Since the majority of hydrophobic groups are located in the core of globular proteins, the binding is related to the denaturation of the protein and the accessibility of these groups to the column functional groups. Proteins can be purified using reverse phase chromatography, but usually must be refolded in some way to regain functionality (i.e. the native state)

Preparation of resins

The steps in preparing a chromatographic resin typically involve:

1. Hydration of resin
2. Decanting fines
3. Equilibrating the resin and preparing a slurry
4. Degassing the slurry

• Resins come either dry or preswollen. If they are dry they need to be hydrated. This is usually accomplished by mixing the dry resin with buffer and letting it hydrate slowly overnight (or faster at higher temperatures
• After the resin has hydrated and settled, very fine particles will settle at the top. These "fines" slow the flow rate of the packed resin. The settled resin is therefore carefully decanted to discard these fines.
• The resin is then equilibrated in the buffer to be used for the analysis. Equilibration usually involves pH'ing the resin, or buffer exchanges. Never use a stir bar when pH'ing the resin (it can mechanically shear the resin and produce fines), rather stir the resin slurry with a stir rod.
• After the equilibrated resin has settled, an equal volume of buffer is added to produce a 50% slurry of resin. This is usually "thin" enough to allow air bubbles to escape when packing the column.
• Finally, the slurry is degassed prior to packing the column. This will help minimize the formation of air bubbles.

Packing the column

Low pressure columns are typically packed using gravity.

• Add a small amount of buffer to the bottom of the column.
• Place a packing reservoir on the top of the column. Since we will be using a 50% slurry we will have a volume which is 2x the column volume and its best to pour the resin in all at one time. Thus, the packing reservoir should have a volume equal to, or greater, than the column volume.
• Carefully pour the resin slurry into the packing reservoir/column, avoiding the introduction of air bubbles as much as possible
• Let the column sit for about 5 minutes to allow large air bubbles to escape
• Open the column valve at the bottom and allow the column to pack under gravity
• Note the top of the resin bed. It will move down as the column packs. When the column is packed the top of the resin bed will no longer move down.

Plumbing

Chromatography systems may be run using only gravity and a beaker to collect the appropriate fraction. Most common systems, however, will include the following:

• A pump. Usually a peristaltic pump with variable flow rate and a communications port for a controller. The pump is usually set up to push buffer through the column, rather than sucking buffer out of the column (which can cause a low pressure condition with production of air bubbles)
• A detector. This is typically a UV (A₂₈₀) detector. Most detectors are of the two-cell type - meaning that you can have plain buffer as a blank in the detector while analyzing your column fractions. The detector sends the absorbance information to a chart recorder to be displayed (printed)
• A fraction collector. This allows you to collect fractions either by number of drops (~30 per ml) or by time. In conjunction with a controllable pump, time collection translates to volume. The fraction collector will typically have an communications port to output a signal when it changes fractions and to receive commands from the detector/controller on some sophisticated systems.
• A chart recorder. This will print a continuous trace of the detector output and the fraction collector event marker (signalling when a fraction changes). Fractions can also be read individually on a UV spectrophotometer if a chart recorder is unavailable.

• If a gravity system is used, a safety loop should be installed to prevent the column from drying up if the buffer is used up when the column is unattended

1998 Dr. Michael Blaber