Section 3.1.5.1. Conventional protein purification

Those of us old enough to remember the task of purifying proteins from their natural sources, using conventional (as opposed to affinity) chromatography, where a 5000-fold purification was not unusual and the purifications routinely began with kilogram quantities (wet weight) of E. coli paste or calves' liver, are most grateful to those who developed efficient systems to express recombinant proteins. In most cases, it is possible to develop expression systems that limit the required purification to, at most, 20- to 50-fold, which vastly simplifies the purification procedure and concomitantly reduces the amount of starting material required to produce the 5–10 mg of pure protein needed to begin crystallization trials. This does not mean, however, that the process of purifying recombinant proteins is trivial. Fortunately, advances in chromatography media and instrumentation have improved both the speed and ease of protein purification. A wide variety of chromatography media (and prepacked columns) are commercially available, along with technical bulletins that provide detailed recommended protocols for their use. Purification systems (such as Pharmacia's FPLC and ÄKTA systems, PerSeptive Biosystems' BioCAD workstations and BioRad's BioLogic systems) include instruments for sample application, pumps for solvent delivery, columns, sample detection, fraction collection and information storage and output into a single integrated system, but such systems are relatively expensive. Several types of high capacity, high flow rate chromatography media and columns (for example, Pharmacia's HiTrap products and PerSeptive Biosystems' POROS Perfusion Chromatography products) have been developed and are marketed for use with these systems. However, the use of these media is not restricted to the integrated systems; they can be used effectively in conventional chromatography without the need for expensive instrumentation.

In designing a purification protocol, it is critically important that careful thought be given to the design of the protocol and to a proper ordering of the purification steps. In most cases, individual purification steps are worked out on a relatively small scale, and an overall purification scheme is developed based on an ordering of these independently developed steps. However, the experimentalist, in planning a purification scheme, should keep the amount of protein needed for the project firmly in mind. In general, crystallography takes a good deal more purified protein than conventional biochemical analyses. Scaling up a purification scheme is an art; however, it should be clear that purification steps that can be conveniently done in batch mode (precipitation steps) should be the earliest steps in a large-scale purification, chromatographic steps that involve the absorption and desorption of the protein from columns (ion-exchange, hydroxyapatite, hydrophobic interaction, dye-ligand and affinity chromatography) should be done as intermediate steps, and size exclusion, which requires the largest column volumes relative to the amount of protein to be purified, should generally be used only as the last step of purification. If reasonably good levels of expression can be achieved, most recombinant proteins can be purified using a relatively simple combination of the previously mentioned procedures (Fig. 3.1.5.1), requiring a limited number of column chromatography steps (generally two or three).

Figure 3.1.5.1 | top | pdf | Protein purification strategy. Purification of proteins expressed at reasonably high levels typically requires only a limited number of chromatographic steps. Additional chromatography columns (indicated in brackets) can be included as necessary. Affinity chromatography can allow efficient purification of fusion proteins or proteins with well defined ligand-binding domains.

All protein purification steps are based on the fact that the biochemical properties of proteins differ: proteins are different sizes, have different surface charges and different hydrophobicity. With the exception of a small number of cases involving proteins that have unusual solubility characteristics, batch precipitation steps usually do not provide substantial increases in purity. However, precipitation is often used as the first step in a purification procedure, in part because it can be used to separate protein from nucleic acids. Nucleic acids are highly charged polyanions; the presence of nucleic acid in a protein extract can dramatically decrease the efficiency of column chromatography, for example by saturation of anion-exchange resins. If the desired protein binds to nucleic acids and the nucleic acids are not removed, ion-exchange chromatography can be compromised by the interactions of the protein and the nucleic acid and by the interactions of the nucleic acid and the column. The most commonly used precipitation reagents are ammonium sulfate and polyethylene glycols. With little effort, the defined range of these reagents needed to precipitate the protein of interest can be determined. However, if the precipitation range is broad, it may be only marginally less efficient simply to precipitate the majority of proteins by addition of ammonium sulfate to 85% saturation or 30% polyethylene glycol 6000. Precipitation can be a useful method for concentrating proteins at various steps during purification and for storing proteins that are unstable upon freezing or upon storage in solution.

Column chromatography steps in which the protein is absorbed onto the resin under one set of conditions and then eluted from the column under a different set of conditions can produce significant purification. Anion-exchange chromatography is usually a good starting point. Most proteins have acidic pIs, and conditions can often be found that allow binding of the protein to anion-exchange matrices. Elution of the protein in an optimized gradient often yields greater than tenfold purification. If conditions cannot be found under which the protein binds to an anion-exchange resin, a reverse strategy can be advantageous. Conditions can be adjusted to promote the binding of most proteins, yielding a flow-through fraction enriched for the protein of interest. Fewer proteins interact with cation-exchange resins; if the desired protein binds, this can be a powerful step. Use of an anion exchanger does not necessarily preclude use of a cation-exchange column; under appropriately chosen sets of conditions (most notably adjustment of pH), a single protein can bind to both resins. Hydroxyapatite resins provide a variation of ion-exchange chromatography that can be extremely powerful for some proteins. While hydroxyapatite columns (traditionally just a modified form of crystalline calcium phosphate) have the reputation of slow flow rates, alternative matrices exhibiting improved flow properties have made hydroxyapatite chromatography significantly less tedious. Hydrophobic interaction chromatography can also provide significant purification and has the advantage that the protein is loaded onto the resin in a high ionic strength buffer, making it a good step following ammonium sulfate precipitation. Proteins can behave very differently with different hydrophobic matrices, and an exploration of a variety of different resins is often a worthwhile exercise. Several tester kits containing an assortment of resins are commercially available. Dye-ligand chromatography can also be explored using an assortment of test columns. Several of the dyes, most notably Cibacron Blue F3GA, have structures that resemble nucleotides and have been useful in purifying kinases, polymerases and other nucleotide-binding proteins. However, many proteins have significant affinity for various dyes, independent of nucleotide-binding activity, and the usefulness of dye-ligand chromatography for any specific protein needs to be determined empirically.

Size-exclusion chromatography, which does not involve absorption of the protein onto the matrix, rarely provides as much purification as the chromatography steps described above. However, this can be a good step to include at the end of a purification scheme. Isolation of a well defined peak in the included volume separates intact, properly folded protein from any damaged/aggregated species that may have been generated during the purification procedure. Furthermore, size-exclusion chromatography can provide a useful indication of whether the protein is a well defined, folded, compact, monodisperse population, or whether it is oligomerizing, aggregating or exists in an unfolded or extended form. Although size-exclusion chromatography does not provide a definitive analysis of such behaviour, migration of the protein consistent with its expected molecular weight is generally a good sign; elution of a relatively small protein in the void volume suggests a need for further analysis. Size-exclusion-chromatography media are available for the fractionation of proteins in many different size ranges. Substantial improvement in purification can be achieved by choosing a size range that is optimal for the protein of interest. However, the ability of size-exclusion columns to separate proteins of different molecular weights is dependent on the amount of protein loaded on to the column. Better purification is obtained when relatively small volumes of protein (generally 1–2% of the column bed volume) are loaded on size-exclusion columns. If really large amounts of protein are needed for a crystallography project, it can be difficult (and expensive) to set up size-exclusion columns large enough to fractionate the desired amount of protein.