Protein Purification:
Classical Approaches











INTRODUCTION
 
 

In Chapter 1 we discussed various approaches to obtaining starting material for the purification of proteins. Now we consider some of the standard approaches in purification. While many proteins are now purified using various types of affinity chromatography (considered in Chap. 3), the classical approaches described in this chapter are still in everyday use, either alone, in systems where the appropriate affinity chromatographic approaches have not been worked out, or usually in conjunction with affinity chromatography.

Before examining these "classical" approaches, however, two important general considerations must be discussed. The aim in protein purification is self-evident: preparation of a "pure" protein. The achievement depends, however, on the definition of purity. With the increasingly sensitive methods of detecting proteins that have been developed in recent years (discussed in detail in Chap. 4 in the section on electrophoresis) it has become considerably more difficult to prepare a "pure" protein. The main question is, pure enough for what purpose? The purity required for the accurate determination of a molecular weight may be quite different from that required for structural studies of the sequence of the polypeptide chain or for enzyme kinetic or ligand binding studies. Thus a pragmatic approach to the question of protein purity must be used, and this is discussed in these and other contexts in subsequent chapters. Since the determination of purity is usually based on one or more of the various approaches used to establish the molecular weight of a protein, further discussion is contained in Chapter 4.
 
 

During the course of protein purification, the specific activity is followed from step to step and a frequently used criterion of purity is the achievement of a constant specific activity for several steps. This approach is particularly useful when constant specific activities are obtained for steps involving quite different physical bases for separation (such as molecular size and ionic properties). It is, however, advisable to determine the purity of the sample independently.
 
  The second important consideration involves the yields from the purification scheme used and the amounts of "pure" protein that are required. For some purposes small amounts of highly purified material are desirable; on other occasions larger amounts may be required and judgments then have to be made as to whether particular steps in a purification scheme which may have low yields but good increases in specific activity are justified. As discussed previously, an informed choice based on a thorough understanding of the pitfalls of a particular experimental: approach can allow the researcher to make such decisions.
 
 

PRELIMINARY FRACTIONATION PROCEDURES
 
 

The approaches that we consider in this section may yield only a few-fold purification; however, their use is not restricted to purification purposes alone. Early stages in most purification schemes have three other motives in addition to increased specific activity:
 
 

 1. The rapid removal of proteolytic enzymes that might otherwise degrade the desired protein. Protease inhibitors may not always be sufficient to block the action of either specific or nonspecific protcases that may be present at the early stages of a purification or may be activated during a purification.

2. The concentration of starting material to more managable volumes. In many of the procedures used, large volumes of material are not desirable: some of the precipitation methods described here are useful for effective and rapid concentration of the starting material-with the added advantage that they yield a purification as well.

3. The removal of material that may interfere with subsequent stages of the purification. In various procedures the desired protein is adhered to an immobile phase to allow contaminating proteins to be washed away. Whether this immobilization is by specific affinity, as in affinity chromatography, or by the general characteristics of the protein, as in ion-exchange or hydrophobic chromatography, it is often necessary to remove as much nonspecific protein as possible first so as to prevent interference with the immobilization.
 
 

Ammonium Sulfate Precipitation
 
 

Differential precipitation of proteins by ammonium sulfate is one of the most widely used preliminary purification procedures. It is based on the differing solubility proteins have in ammonium sulfate solutions and can result in a two- to fivefold increase in specific activity (in the case of glyceraldehyde-3-phosphate dehydrogenase from rabbit muscle, essentially homogeneous protein can be prepared simply by using a three-step ammonium sulfate precipitation procedure). Provided that appropriately buffered ammonium sulfate solutions are used to protect the desired activity, recoveries approaching 100% can be expected. A typical protocol (as outlined in Fig. 2-1) consists of adding ammonium sulfate to give a certain percentage saturation, followed by a period of time for proteins to precipitate and a centrifugation step to collect the precipitate.

Once it is known in what range of ammonium sulfate concentrations the desired protein precipitates, the initial solution can be adjusted to a concentration sufficiently below this so that none (or very little-there are always judgments to be made) of the desired protein precipitates and the undesired protein can be removed by centrifugation. The ammonium sulfate concentration is then raised to a level sufficient to precipitate all (or most) of the desired protein while leaving in solution other undesirable proteins, and the precipitate retained for further purification. The appropriate concentration ranges are conveniently ascertained by screening a range of concentrations for small samples and determining the activity of the desired protein in the supernatant after centrifugation. Once this has been done, the appropriate concentration ranges can easily be chosen. It is important that when scaling up the total protein concentration in the sample is similar to that in the trial since the solubility of most proteins in ammonium sulfate is quite dependent on the total protein concentration.
 
 

Isoelectric Precipitation
 
 

Essentially similar in practice to ammonium sulfate precipitation, this approach is based on the fact that most proteins precipitate when there is no overall charge on the molecule-that is, at the isoelectric pH-since charge-charge repulsions tend to keep proteins in solution. Because proteins in general have fairly unique isoelectric points this procedure can give good, quick separation of unwanted proteins. In practice, the pH dependence of the stability of the desired protein can be a determining factor in the method's usefulness. Some limitations exist for the effective concentration of proteins depending on how readily the desired protein, once precipitated, can be redissolved. A variation of this procedure involves the pH dematuration of unwanted proteins and their removal by centrifugation, an approach that can be assisted by factors that affect (increase) the pH stability of the desired protein. Substrates or other ligands may increase stability to, for example, low pH, thereby allowing a lower pH to be used than would otherwise be the case. As with ammonium sulfate precipitation procedures, the appropriate conditions are established on a small scale.

 

Solvent Precipitation
 
 

As with isoelectric precipitation, solvent precipitation can be used in two basic ways. Ethanol or other organic reagents, by changing the dielectric constant of the solvent, frequently induce precipitation of proteins that can then be collected and treated in ways similar to those described for ammonium sulfate precipitation.

Polyethylene glycol, of a variety of polymer sizes, is commonly used in fractional precipitation procedures. Any of the readily soluble polyethylene glycols can be employed: Those of higher molecular weight are frequently useful in concentration schemes. The dilute protein solution is placed in dialysis tubing and surrounded with dry polyethylene glycol, which absorbs water through the semipermeable membrane and concentrates the dialysis tube contents.

In the second approach, unwanted proteins in a mixture might be specifically inactivated and denatured by an organic solvent, thus allowing the contaminating protein to be removed. During the purification of Jack Bean ot-mannosidase, contaminating fl-N-acetylhexosaminidase is removed by specific inactivation with pyridine followed by centrifugation of the precipitated contaminant.
 
 

Heat Precipitation
 
 

Finally, we consider precipitation of contaminating proteins by heat denaturation. Different proteins have different stabilities at elevated temperatures, and if the desired protein has a greater heat stability than contaminating proteins, incubation at elevated temperatures for periods of time varying from a few minutes to a few hours often precipitates unwanted proteins, which can then be removed by centrifugation. As with pH-induced denaturation, the stability of the desired protein at elevated temperatures may in some cases be enhanced by the presence of substrates or other specific ligands.
 
 

CHROMATOGRAPHY METHODS

Gel Filtration

 

A large variety of gel filtration media are available and all work primarily on the basis of an exclusion limit, which is generally defined as the protein size that cannot penetrate the bead space of the material and thus is excluded from the column matrix. Proteins larger than this size co-chromatograph through the column and elute in the void volume (Vo) of the column. Other material falls into two classes:
 
 

1. Material smaller than the exclusion limit which does not physically interact with the matrix material. Such material can be considered as having "normal" gel filtration behavior, and its elution volume (Ve) depends on the size of the material relative to the pore size of the matrix.

2. Material that interacts with the matrix material. Any physical interaction (causes of which are considered in the context of the nature of the matrix material) causes a retardation of the chromatographed material greater than what would be expected to occur simply by its ability to penetrate matrix space, and thus such material elutes at an anomalous elution volume. If the interaction with the matrix is of sufficient magnitude, the interacting material may elute at a volume larger than the normal total elution volume (Vt) of the column, which is the volume taken to elute molecules from the column having sizes similar to the bulk solvent volume.
 
 

Experimental Determination of Chromatography Parameters. The three parameters Vo Ve and Vt are used to describe the behavior of a particular molecule on a gel filtration column and must be determined experimentally. Three types of chromatography experiments can be envisaged:
 


1. In the ideal case, the sample size loaded onto the column is very small compared to the volume of the packed matrix material. In this instance (Fig. 2-2A), the elution volume, Ve is simply the volume of eluent collected from the start of loading the sample to the midpoint of the sample elution.

 2. When the sample size is not negligible compared to the bed volume of the column, the elution volume is usually calculated from the midpoint of the sample loading to the midpoint of the elution profile (Fig. 2-2B).

3. If the sample size is so large that a simple elution peak is not obtained (Fig.2-2B, the elution volume is calculated from the start of the sample loading to the midpoint of the ascending side of the elution profile.

We should consider practical limits to the sample size that can be used in gel filtration chromatography. The separation volume (Vsep) between two peaks A and B can be defined as

                                            Vsep = VeB - VeA                        (2-1)

If a sample eluted from a column behaved ideally, the maximum sample size could be as great as Vsep. However, as the sample size is increased the size of the eluted peaks increases, and for resolution of the peaks the sample size should always be smaller than Vsep.

 One problem is that under many experimental situations Vsep is not known, so as a general rule the sample size should be kept as small as is practical-in the range 2 to 5% of the column bed volume. For desalting applications, however, where the matrix has usually been chosen such that the desired protein elutes in the void volume while the elution volume of the "salt" approaches the total column elution volume, it is possible to use much larger sample sizes-in the region of 20% of V -and achieve effective desalting with minimal sample dilution.

The total volume, Vt is usually obtained by loading a sample containing a small molecule (which does not physically interact with the matrix material) that can be conveniently monitored by absorbance or radioactivity and directly determining its elution position as described previously for Ve, estimations.
 
 

The void volume of the column is determined similarly but by using a sample containing a macromolecule of sufficient size such that it is totally excluded from the matrix. In many instances blue dextran is used, although for columns employed to separate smaller molecules, a protein such as BSA is often convenient. As mentioned, the elution position of the void volume material is obtained as described for Ve determinations. Several other parameters can be defined and estimated once Ve, Vt, and Vo are known.

The elution of a solute molecule in gel filtration chromatography can be characterized by a distribution coefficient, kD:

                                kD = {Ve - Vo }/ Vs                                 (2-2)

where Vs is the volume of the stationary phase, which is the volume of solvent that can permeate the matrix and is accessible to small molecules (those which elute at Vt

                                Vs = Vt - Vo - Vgelmatrix                                    (2-3)
 
 

In practice Vs is difficult to determine and is usually approximated by V, - VO, and kD is replaced by Kav

                             Kav = [Ve - Vo]/[ Vt - Vo]                             (2-4)

 and is not a true partition coefficient. These parameters are summarized in Fig. 2-2).

Prior to considering the chemical nature of the various matrix materials available, we should discuss several other choices that have to be made concerning the practical setup of a gel filtration experiment. Many of the available matrices come in different particle sizes, from superfine to coarse. The smaller particles of the superfine grades give better physical packing of the matrix than does larger material, resulting:in less zone broadening of peaks and consequentially, better resolution. The larger, particle grades have considerably faster flow rates, however, which may be advantageous in working with unstable material or with rapid procedures such as desalting. The physical size of the column must also be chosen: Since the resolution of separated peaks increases as the square root of the column length, long columns in general give better separation than short columns but elute more slowly. The diameter of the column is important since narrow columns can hinder ideal passage of solvent through the column and wide columns give increased sample dilution. By far the most important choice regards the sample viscosity: High sample vicosity leads to distortion of elution peaks, which vary with the molecule size. The sample and buffer viscosity should not differ by more than a factor of 2, which, for most proteins, puts an upper limit for concentration of 50 to 70 mg/ml. It must be emphasized that many proteins undergo concentration-dependent aggregation, which can lead to anomalous gel filtration behavior not just due to viscosity problems, but also because of the molecular weight and size polydispersity that such a phenomenon can create.
 
 

Choice of Gel Filtration Matrix Material. Three basic types of matrix material have been used which differ somewhat in their physical and chemical properties. The most common are the cross-linked dextrans (e.g., Sephadex). This bead-formed gel is prepared by cross-linking dextran with epichlorohydrin. The resultant gel contains a large number of hydroxyl groups, which makes it quite hydrophilic and causes the gel to swell readily in water or electrolyte solutions. The porosity of the gel, and hence the useful fractionation range, is governed by the degree of cross-linking.
 
 

As discussed earlier, adsorption of material being chromatographed to the matrix leads to anomalous elution. Two principal types of adsorption must be considered: ionic and aromatic. With the cross-linked dextrans these effects are particularly noticeable on the highly cross-linked gels used to fractionate small molecules. The matrix material contains a low level of carboxyl groups, which at low ionic strength lead to the retardation of positively charged species and increased exclusion of negatively charged species. At ionic strengths above about 0.02, however, these effects become negligible with most proteins or peptides. A variety of aromatic compounds (such as purines, pyrimidines, dyes, and hydrophobic peptides) interact with the matrix material, causing additional retardation. These interactions can be suppressed by using urea or phenol-acetic acid-water buffer systems for elution. However, such interactions are not always undesirable. Frequently, fairly similar aromatic compounds can be separated by making use of their interactions with the matrix, which can be modulated by changing the composition of the elution buffer. The addition of methanol or ethanol tends to increase the strength of these interactions, while altered ionic strength or pH can be used to weaken them. In essence this is hydrophobic chromatography, which is discussed in more detail later in this section.
 
 

The second type of matrix material commonly used consists of allyl dextran cross-linked with N,N'-methylene bisacrylamide, which gives a quite rigid gel structure having well-defined porosity. Due to the rigidity of the matrix, this type of material (e.g., Sephacryl) can easily be used with organic solvents with a much smaller effect on pore size (and hence distortion of the fractionation range) than with the Sephadex-like matrices. In general, the Sephacryl-like resins give better flow rates for equivalent fractionation ranges, but are only available for the separation of larger molecules [20,000 - 106 daltons (Da)].
 
 

Because of its high matrix density (and consequent carboxyl group density) these matrices have more pronounced ionic adsorption properties than the simple dextrans. In general, higher-ionic-strength buffers are therefore used with this type of material to help suppress such effects.

Finally, various derivatives of agarose have been used. The gel structure of agarose-based gels is stabilized by hydrogen bonding rather than chemical crosslinking but is quite stable under most conditions. The porosity is governed by the concentration of agarose in the material. The open structure of the agarose-based matrix makes this type of material (e.g., Sepharose) most suitable for the fractionation of very large macromolecules, although matrices with high agarose contents (up to approximately 6%) can be used with proteins in the range 10,000 Da and upward.

Such resins do contain low levels of carboxyl and sulfate groups, which can cause retardation of basic proteins, although as discussed for the other resins, these effects can be minimized by using elution buffers of reasonable ionic strength (I > 0.02). The thermal and chemical stability of agarose gels can be increased (with negligible effect on porosity) by chemical cross-linking with 2,3-dibromopropanol. The enhanced stability of the resultant material allows alkaline hydrolysis (under reducing conditions) to be used to remove sulfate groups, giving a gel with a very low content of ionic groups and consequent elimination of most ionic adsorption effects. The basic structures of these various resins is given in Fig. 2-3.

Ion-Exchange Chromatography

 Ion-exchange chromatography is based on the simple concept that at a given pH most proteins have a charge (either overall negative or positive, depending on the pI of the protein) and hence are attracted to (i.e., interact with) an opposite charge. Different proteins have differing amounts of charge and hence adhere more or less tightly to the opposite charge compared to other proteins. 'This interaction causes a retardation in chromatography provided that the matrix material has the appropriate charge. In essence, the various matrices we have discussed for gel filtration chromatography are the basis of ion-exchange matrices: The matrix is derivatized to give it the desired anion- or cation-exchange properties. The commonly used functional groups are shown in Fig. 2-4. The basic properties of the support matrix are as discussed previously and should be selected based on the size of the proteins to be fractionated. If it is necessary to use polar organic solvents, the matrix should be of the chemically cross-linked agarose type.

Once the appropriate resin has been chosen (more about this later) only the ionic strength and pH of the loading buffer need to be considered. Since the interaction of a protein with the matrix is through charge-charge, ionic strength of the loading buffer should be kept low to maximize interaction. The capacity of the column to bind the appropriately charged species is dependent on the number of oppositely charged groups available, which in turn depends on the pK values of the groups and the pH of the medium. Figure 2-5 shows titration curves for some of the commonly used ion exchangers. DEAE-based resins indicate the presence of multiple charged groups but have good capacity below a pH of about 8.5 (the pK of the normal DEAE group is about 9.5). If an anion-exchange resin is needed at higher pH, the QAE-type resins (pK around 12) can be used at significantly higher pH values. Similar considerations apply to the cation exchangers CM- (pK around 3.5) and SP- (pK around 2.0).

Elution of material from an ion-exchange matrix is generally achieved in one of two ways. The ionic strength of the elution buffer is raised to a level that decreases the charge-charge interaction of the chromatographed material with'the matrix, or the pH of the eluent is changed so that the charge of the adhered protein is altered such that it no longer interacts with the matrix. The pH must be decreased with anion-exchange material but increased with cation-exchange material. In some cases a combination of these two effects is used. The change is usually produced by running a gradient of increased-ionic-strength buffer (or the appropriate pH gradient) through the column and monitoring the eluent for protein, activity, and so on, to locate the desired protein. Separation is achieved at two levels: First, not all proteins adhere to the column during the adsorption phase of the experiment. Second, as the elution gradient proceeds, different proteins elute based on the avidity of their interaction with the matrix; weakly bound proteins (i.e., those with the lowest charge density under the initial adsorption phase) are eluted first, while highly charged proteins require more drastic changes in pH or ionic strength.
 
 

Determination of Adsorption and Elution Conditions. During the initial stages of establishing a protein purification it is necessary to establish: (1) what type of ion exchanger should be used, (2) what conditions are necessary for adsorption, and (3) what conditions are necessary for elution. In general, conditions where the wanted protein adheres to the matrix should be established rather than conditions where other proteins adhere but not the wanted protein, since in the former case separation is achieved at both the loading and elution stages. In the absence of prior knowledge about the molecular properties of the protein it is convenient to screen a wide range of pH values rapidly with a particular resin type using the simple mixing and centrifugation procedure outlined in Fig. 2-6. Activity measurements on the supernatant allow one to establish adsorption (and, of course, elution) conditions rapidly.

An alternative approach for establishing optimal separation conditions for closely related molecules such as lactate dehydrogenase isoenzymes involves electrophoretic titration curves. This depends on the normal charge on the protein and its isoelectric point (pI). Electrophoresis is carried out in a vertical plane using a.large-pore gel matrix such as agarose or a low-percentage acrylamide which has a preformed horizontal pH gradient generated from the appropriate ampholines. As indicated in Fig. 2-7, the sample containing the mixture of proteins is added to a central horizontal well and electrophoresis is begun.

During electrophoresis the proteins move either toward the cathode or the anode or, if the pH is at their isoelectric point, they do not move at all. The rate of movement depends on the pH relative to the pI of the proteins. After electrophoresis is terminated the proteins are stained (for activity if appropriate; see later) and the titration curves examined. A typical set of @ curves for lactate dehydrogenase isoenzymes is shown schematically in Fig. 2-8. From the results the pH that gives the largest separation on the basis of charge can easily be evaluated. This pH gives optimal separation during elution from the appropriate ion-exchange resin.

In addition to being a suitable purification procedure for many proteins, ionexchange chromatography has a number of other attributes that are outlined in Table 2-1. Particularly useful are the potential concentration of a wanted protein during a

purification procedure and the removal of metals from metalloproteins during the preparation of apoenzymes. In protein concentration it is often convenient to use a stepwise elution procedure rather than gradient elution.

Although we have discussed gel filtration and ion exchange in terms of column chromatography, both approaches are readily adaptable to thin-layer chromatography, which is particularly useful when a two-dimensional separation involving electrophoresis in addition to gel filtration (for example) is used. Ion-exchange methods are also particularly suitable for batchwise procedures since nonadsorbed material can easily be removed by washing and centrifugation prior to elution.
 
 

Hydrophobic Chromatography
 
 

Although the use of hydrophobic chromatography in protein purification has been popularized only recently, the idea owes its genesis both to gel filtration and affinity chromatography (Chap. 3). The matrix employed is usually based on agarose that has been derivatized in aprotic solvents with epoxides (which have relatively large alkyl chains). A generalized formula for the derivatives is

                        agarose-0-CH2-CH[OH]-CH2-0-R                         (2-5)

where R represents the alkyl chain and usually contains between 5 and 12 carbons. Any protein with some external hydrophobic characteristics tends to interact with such a matrix and be retarded relative to proteins lacking such characteristics. In general, the capacity of such columns for protein increases with increasing hydrophobicity of the substituent, with increasing degree of substitution, and with increasing ionic strength. The latter characteristic is quite distinct from the charge-charge interactions described earlier for ion-exchange chromatography, and leads to the principal method of elution from such a matrix: The ionic strength of the loading buffer is kept high and elution is achieved using a decreasing-ionic-strength gradient. Because the porosity of the matrix is decreased as the hydrophobicity of the substituent increases, generally a lower degree of substitution is employed, which is compensated for by using a higher initial ionic strength to maximize capacity and adsorption. In circumstances where adsorption is particularly tight (i.e., long alkyl chains, high degree of substitution, high ionic strength), complete desorption of adhered protein is sometimes difficult to achieve by decreased ionic strength alone. In such cases the addition of glycerol or ethylene glycol to the elution buffer tends to enhance desorption. In some instances increased desorption can be achieved by adding to the elution buffer ligands specific for the wanted protein that bind and change the conformation to one with lower external hydrophobicity. The converse of this situation-a ligand that upon binding leads to increased hydrophobicity can be used to increase adsorption of the wanted protein to the matrix. In such an instance elution would be enhanced by omitting the ligand from the elution buffer.
 
 

High-Pressure Liquid Chromatography;

High-Performance Liquid Chromatography

 

Both terms above are represented by "HPLC," and over the last five years these techniques have become increasingly useful in the isolation and characterization of molecules of biological interest whether, in the context of this book, they are proteins, peptides, or amino acids. HPLC is a philosophy rather than a particular technique, and in fact under the term "HPLC" fall each of the chromatographic techniques we have discussed so far, together with affinity chromatography. The fundamental principles remain the same whether used in conventional column Chromatography or in HPLC methods and are not reiterated here, although some aspects of reversephase HPLC (which is derived from hydrophobic chromatography) are amplified since at present this is the most commonly used of the HPLC techniques.
 
 

In general, each of the approaches employs an immobile phase bonded onto a porous silica, which allows high flow rates to be used, and a mobile phase, whose composition is appropriate for the particular technique. We now briefly consider some of the characteristics of each of the HPLC techniques.
 
 

1. Gel Filtration Chromatography. A variety of bonded phases have been used to cover the cationic surface of silica and prevent nonpermeation effects. These include glycerylpropyl, diol, and N-acetylaminopropyl silane. Although a number of non-silica-based support materials have been used, most work has involved the silica-based material.
 
 

2. Ion-Exchange Chromatography. Again, silica supports with an associated immobile phase of, for example, polyethyleneimine have produced column packing with good stability and high (in the context of HPLC capacity. A variety of organic polymer supports such as polystyrene have also been used, but primarily for lowmolecular-weight molecules.
 
 

3. Reverse-Phase Chromatography. This technique uses reversed phases such as octadecylsilyl (C18), Octylsilyl (C8), butylsilyl (C4), and propysilyl (C3) bonded to silica supports. RP-HPLC is essentially derived from hydrophobic chromatography and is probably the most widely used of the HPLC techniques, having found applications in both purification and characterization of proteins, peptides, and peptide mixtures such as might be obtained by proteolytic digestion of a protein. In general, the retardation of a molecule in RP-HPLC depends on no one parameter such as size or charge, although there is an approximate correlation between retention time (i.e., elution time) and the percentage of hydrophobic residues in the protein or peptide, although conformational effects often distort this relationship. Two types of elution are frequently used in RP-HPLC.
 
 

(a) Isocratic Elution. The composition of the mobile phase is kept constant (this phase usually contains an organic solvent such as acetonitrile and an aqueous solvent such as trifluoroacetic acid or phosphoric acid). With isocratic elution the composition of the mobile phase must be predetermined since the retention time of a protein changes with its composition, especially in reference to the context of the organic solvent.
 
 

(b) Gradient Elution. Because of the sensitivity of retention time to the content of the organic solvent, proteins and peptides are usually eluted with an acidic mobile phase using a gradually increasing organic solvent content.
 
 

ELECTROPHORETIC METHODS

 

Two electrophoretic methods are available that can conveniently be used in general protein purification. These are native polyacrylamide gel electrophoresis (PAGE) and isoelectric focusing. Both depend on the movement of proteins through a matrix support on the basis of the charge of the native protein. In native PAGE, the rate of movement is governed by other factors, such as the porosity of the gel and the molecular weight and the shape of the protein, all of which are discussed in detail in Chapter 4 in the context of molecular weight determination. In isoelectric focusing the distance of movement is governed by the isoelectric point of the protein, and the rate of movement is less important since the experiments are continued until equilibrium is reached. In principle, both techniques are somewhat similar: a mixture of proteins is separated electrophoretically and the desired protein is identified on the electrophoretogram and eluted from the support material.
 
 

Detection of Active Material after Electrophoresis

 

There are three basic procedures that can be used for the detection of native material after electrophoresis: (1) measurement of enzymatic activity; (2) detection by specific ligand binding, using, for example, a fluorescent or radioactive ligand; and (3) detection by antibody binding if a suitable antibody to the native protein is available. These procedures can be performed directly in the separating matrix, or after the separated material has been transferred to a more suitable matrix for such detection, via a process known as "blotting." Although detection of active proteins directly in the separating matrix can in theory be achieved, a number of factors may result in such a procedure being ineffective or undesirable. Substrate or ligand diffusion through the matrix may limit the sensitivity or success of direct staining. The desired protein may have little or no direct activity while constrained within the rigid separating matrix. The problem of substrate or ligand diffusion can be overcome by using ultrathin support material; however, the latter problem remains a potential pitfall. These difficulties are largely overcome by the process of blotting. Blotting is gradually replacing the more laborious but still quite effective process of slicing the matrix material into small pieces, eluting protein with a suitable buffer for subsequent enzymic analysis, and then assaying the eluted material for its specific activity.
 
 

Nitrocellulose paper is the most widely used material for blotting since most proteins adhere to nitrocellulose, and such papers have reasonable capacity, making subsequent detection more facile. Under conditions for transfer both the nitrocellulose paper and the protein are probably negatively charged, and hydrophobic rather than ionic effects are probably involved in protein-paper binding. The major problem with nitrocellulose is that low-molecular-weight proteins may bind with low affinity and as a result be washed from the paper during subsequent handling. Alternative types of matrix involving covalent immobilization of blotted proteins can be useful; however, such a process may inactivate the protein.
 
 

Three principal ways exist for transferring proteins from the separation matrix to the detection matrix in blotting. In the simplest, the separation matrix is sandwiched between two sheets of nitrocellulose filter paper and the sandwich completed with appropriate support material, then placed into a chamber containing buffer and transfer by simple diffusion takes place. Although slow, such a procedure can be quite effective and provided that denaturation of the desired protein does not occur during the transfer, is simple, cheap, and effective. The second procedure is a variant of the first, where mass flow of solvent is induced through the gel and the nitrocellulose paper. The gel is placed in a buffer reservoir, the nitrocellulose paper placed on top, and a stack of absorbing material (such as paper towels) placed on top of the filter paper. This leads to buffer being drawn through the gel and filter paper, resulting in elution of the proteins from the gel and their immobilization on the nitrocellulose paper. The third method involves additional apparatus but can be quite fast and effective. It is based on the electroelution of the sample from the separating matrix onto the nitrocellulose paper. This is possible because proteins adhere to nitrocellulose even in low-ionic-strength buffers. The original gel and nitrocellulose paper are sandwiched together with porous support material and placed into a tank containing a transfer buffer and electrodes. In electroelution proteins of different charge and molecular weight "elute" at different rates, which can present a problem. As an attempt to counter this, PAGE is done with a reversible gel cross-linker such as N,N'-diallyltartardiamide in place of bisacrylamide. The gel is depolymerized, in this instance by incubation with 10 mM periodate for 30 minutes at 22'C, prior to being placed in the sandwich used in electroelution. The inclusion of low concentrations of detergent such as 0. 1 % SDS in the transfer buffer also facilitates electroelution and does not appear to affect the adherence of protein to nitrocellulose (it may, however, in some cases have adverse effects on protein activity or stability).
 
 

Detection of Specific Proteins after Electrophoresis

 

As indicated earlier, it is possible, with gel electrophoresis systems where the native structure of the protein is retained, to stain for specific proteins in situ in the electrophoresis matrix. This can be accomplished by one of three approaches.
 
 

Enzymatic Activity Stains. A variety of staining procedures making use of the
 
  catalytic reaction of the specific protein have been developed. There are two approaches: (1) either a fluorescent substrate or product is followed, and after reaction the loss or appearance of fluorescence is monitored, or (2) the enzymatic reaction product is coupled to a chemical reaction that produces a colored dye located in the gel at the site of the specific protein. Examples of these types of staining procedures are the detection of esterases or dehydrogenases, both shown schematically in Fig. 2-9.

 Detection by Specic Ligand Binding. In a number of cases a specifically bound ligand, either fluorescent or radioactive, can be diffused into the gel and after washing to remove background labeling is located by fluorescence or auto radiography. An extension of this approach is the use of the fluorescent dye ANS to bind proteins in gels run in the presence of denaturants. ANS binds to most proteins with an enhancement of fluorescence, and protein bands can be detected by this. Background effects are minimized in the general gel matrix by the fact that the denaturants tend to quench the fluorescence of free ANS.  

Detection by Binding Fluorescently Labeled Proteins to Specific Target.

 

Any protein that shows a specific interaction with a desired target protein and can be fluorescently labeled can also be used to detect the specific protein in a gel matrix. Usually, the protein is labeled with fluorescein isothiocyanate (the method could also be used with radioactively labeled protein and autoradiography), which is then diffused into the gel matrix, and following destaining the specific interaction is detected by location of fluorescence after exposure to long-range ultraviolet (UV) light. A variety of such stains have been developed using fluorescently labeled antibodies of the appropriate specificity, as well as using fluorescently labeled lectins for the detection of specific glycoproteins. Lectins in particular can be quite useful in this type of approach, as general glycoproteins can be labeled using lectins with little sugar specificity such as concanavalin A, while glycoproteins with specific terminal sugar residues can be labeled using lectins having defined precise specificities (many of which are given in Chapter. 3).
 
 

 Finally, we must consider briefly pertinent aspects of native PAGE and preparative isoelectric focusing. In native PAGE the separating gel is usually topped with a spacer gel of increased porosity that is used to concentrate the sample at the top of the separating gel. They are usually run at fairly high pH (around 8.3), where most proteins carry a net negative charge; however, occasionally a protein might be encountered that electrophoreses away from the gel. Electrophoresis can still be achieved, either by changing the pH or reversing the polarity of the electrodes.
 
 

In preparative electrophoretic techniques the major difficulty after separation and detection of the separated material is the quantitative recovery of the desired protein from the ' matrix. In most cases this can be achieved rapidly and quantitatively by preparati vescale electroelution using an apparatus of the design shown in Fig. 2-10. Because of the multiple membrane construction of such a device it can be used to separate proteins from SDS and from native or isoelectric focusing gels, and quite effectively concentrates the eluted protein.

Although analytic isoelectrofocusing is usually run in either tube gels (infrequently) or in slab gels (more usually), preparative isoelectric focusing is conveniently performed using a flat bed of Sephadex as the ampholine carrier. Large quantities of protein can be handled, and after detection of activity, the desired protein can readily be eluted by scraping the appropriate region of the matrix from the bed, packing it into a column, and eluting as in any gel filtration experiment.
 
 

CHROMATOFOCUSING

 

Chromatofocusing combines the high resolution of isoelectric focusing separations with the high capacity of ion-exchange column chromatography. As in isoelectric focusing, the approach depends on the generation of a pH gradient. Since the charged group on an ion-exchange resin has a buffering action at a particular pH, it will, if eluted with a second buffer at a different pH, form a pH gradient. If proteins are bound to the ion-exchange resin, they elute as the generated gradient reaches the isoelectric point. Optimal resolution by the pH gradient is generally through linear gradients, which are achieved by ensuring that the eluting buffer and the ion exchange resin have constant buffering capacity over the necessary pH range.
 
 

When a protein and eluting buffer first enter the column, the protein either adheres to the matrix of an anion exchange resin if the pH of the eluting buffer is initially higher than the pI of the protein, or it travels down the column if the pI is greater than the initial pH. As the eluting buffer travels through the column, its pH increases until it is greater than the pI of migrating proteins, at which point the formal charge on the protein reverses and it adheres to the matrix. As the pH gradient develops (with an anion-exchange resin an increasing hydrogen-ion gradient is used-that is, the pH decreases as the elution proceeds), the pH drops below the pI of the protein, which is therefore released from the resin and eventually elutes from the column at its isoelectric point. The initial migration of a protein through the matrix, followed by adsorption and release, results in a focusing effect for particular proteins.
 
 

SOME EXAMPLES OF PURIFICATION PROCEDURES

 

There is probably no such thing as a typical protein purification; proteins behave differently in each of the approaches we have discussed, and no individual protein purification of necessity uses all of them. As emphasized earlier, a variety of judgments must be made; sometimes yield will be sacrificed for purity, sometimes a step with a good yield or purification will be omitted for reason of speed required with an unstable protein. The figures and tables on the next few pages show several "typical" purifications, with some comments on the choices for the various steps used.
 
 

There are, however, some overall principles that can be followed as a guide to setting up a purification scheme. After considerations such as the rapid removal of proteases and the concentration of the sample, both of which are often achieved via a precipitation approach, it is usually advisable to employ a technique that is as selective as possible as early as possible. Since such a technique is early in the purification, it should have a high capacity. In general, succeeding stages should use different separating techniques and chromatographic steps should be linked to minimize handling. Ion exchange can precede hydrophobic chromatography since the high ionic strength used to elute proteins from ion-exchange resins gives optimal conditions for adsorption onto hydrophobic matrices. Steps involving dilution (such as gel filtration) should precede steps that increase concentration (ion-exchange chromatography), so that time and effect are not lost in concentrating the sample without benefit of purification.
 
 

High-resolution techniques should be used toward the end of a procedure since these tend to use small sample amounts and may be interfered with by contaminating proteins that can readily be removed during earlier stages.
 
 

Purification of a Metalloendoproteinase ftom Mouse Kidney
 
 

The endoproteinase activities in homogenates and at various stages of the purification were estimated using azocasein as substrate. Azocasein contains dye molecules covalently attached to amino acid side chains in the protein. When the protein is proteolytically degraded, dye-containing peptides are released. These are soluble in 4% trichloroacetic acid (TCA) while the parent molecule precipitates. The dye is quantitated by absorbance measurements on TCA-soluble material at various times of incubation.
 
 

1. Initial homogenates obtained using a Dounce homogenizer were centrifuged at 600g for 10 min at 4oC to give a supernatant (kept) and a residue that was rehomogenized and the second supernatant combined with the first prior to centrifugation at 100,000g. The sediment was resuspended.
 
 

2. Attempts to solubilize activity from 100,000g sediment with various salt concentrations or with mild detergents (e.g., 0.1% Triton X-100 or Brig-35) were unsuccessful and treatment with toluene-trypsin was necessary. This disrupts the membrane and proteolytically removes intrinsic membrane proteins, releasing membrane-bound proteases.
 
 

3. Stepwise ammonium sulfate fractionation was used. Twenty and forty percent caused protein precipitation, but the endoproteinase activity remained in the supernatant. Activity precipitated when the supernatant from the 40% step was raised to 80% saturation. Further increases in ammonium sulfate concentration produced no further activity precipitation.
 
 

4. Precipitated activity was redissolved and dialyzed against 20 mM Tris/HC1 pH 7.5, which was the starting buffer for DEAE chromatography. After loading activity onto DEAE, the column was washed with 50 ml of starting buffer and activity eluted with a NaC1 gradient as shown in Fig. 2-11.

5. Fractions containing more than 300 units of activity per milliliter were pooled, concentrated, and subjected to gel filtration on Sephadex G-200, which gave two peaks of activity (Fig. 2-12).
 
 

6. The first peak appeared pure when it was rechromatographed on Sepharose 6B: the activity eluted as a single symmetrical peak of constant specific activity.
 
 

The second peak had essentially identical enzymatic properties, but as shown in Table 2-2, has a significantly lower specific activity.

 

Reference: R. J. Beynon, J. D. Shannon, and J. S. Bond, Biochem. J., 199, 591-598(1981).
 
 

Purification of an Endogluconase from Clostridium thermocellum

 

Often when proteins that are secreted by bacteria are being purified there is a very large volume of material to be handled. In this particular purification the cells were removed from the medium in late exponential phase by centrifugation at 10,000 for 30 minutes. An alternative approach employed in some cases is to culture the bacteria inside dialysis tubing. A large amount of culture fluid is used outside the tubing to ensure a fresh supply of nutrients and the removal of small molecules that might slow growth. The secreted proteins are, however, retained (together with the cells) within the much smaller volume of the dialysis tubing. The following procedure is summarized in Table 2-3.

1. Although the initial cell-free supernatant was first concentrated by ultrafiltration, ammonium sulfate precipitation was also used to concentrate the large volumes, and was achieved by adding 80% ammonium sulfate and collecting the precipitate.
 
 

After the precipitated material was redissolved the buffer was equilibrated with the start buffer for the DEAE step by continuous ultrafiltration. In this process fresh buffer was repeatedly added to the ultrafiltration cell until the conductivity of the dialysate matched that of the start buffer. The material was then adsorbed onto a DEAE column.
 
 

2. The first DEAE column was eluted with a step gradient using the indicated concentrations of ammonium acetate (Fig. 2-13). Two major areas of activity were obtained-indicated as pooled fractions I and III. Rechromatography of fraction I yielded further purification but still two main peaks of activity resulted, as shown in Fig. 2-14.



3. The major peak, Ila, was selected for further purification on SP-Sephadex, which was eluted with a sodium chloride gradient (Fig. 2-15).
 


4. The single major peak of activity was pooled and subjected to preparative gel electrophoresis on an 8% separating gel. This gave a major peak containing most of the activity and a minor peak with similar specific activity.
 
 

Reference: T. K. Ng and I G. Zeikus, Biochem. J., 199, 341-350 (1981).

 

Purification of Uroporphyrinogen Decarboxylase from Human Er throcytes Y

 

Uroporphyrinogen decarboxylase is a cystolic enzyme that decarboxylates a variety of porphyrinogens. In the purification described here cytosol was obtained from erythrocytes. The enzyme is present in tissues such as liver and in such a case, simple homogenization, in the appropriate buffer to disrupt the cells, releases the cytosol, which is obtained as the supernatant of an initial centrifugation step. The following procedure is summarized in Table 2-4.
 
 
 
 

1. After washing the erythrocytes in saline, hemolysis was achieved by resuspending in ice-cold water for 2 hours at 4'C.
 
 

2. After hemolysis the pH was adjusted to pH 7.0 by addition of 2 volumes of 4 mM phosphate. Hemoglobin was removed by adsorbing activity to DEAE at pH 7.0 in a batchwise procedure. After the resin was obtained by centrifugation it was washed to remove hemoglobin, and activity was eluted by adding 0.5 M KCl.
 
 

 3. Ammonium sulfate precipitation with 25% (w/v) removed some protein, but not activity. Further ammonium sulfate addition (10%, w/v) precipitated the activity, which was then further fractionated using Sephacryl S-200. Active fractions were identified and pooled if they contained >1/3 the activity of the peak fraction. This protocol optimizes the purification achieved in this step. A greater yield could have been obtained by pooling all activity-containing fractions, but purity would have been considerably reduced.

4. Enzyme activity was concentrated by ammonium sulfate precipitation and resuspension in 50 mM phosphate buffer, diluted with an equal volume of 2 mM phosphate containing 2 M ammonium sulfate, and subjected to hydrophobic chromatography on a phenyl-Sepharose column. The column was eluted with a decreasing ammonium sulfate gradient, as shown in Fig. 2-16. The activity eluted just after the protein peak and the fractions with highest specific activity were pooled and subjected to preparative electrophoresis, using a 6.2% separating gel, which produced a further three-fold increase in purification. Activity was recovered from the gel by slicing and eluting with phosphate buffer.
 
 

Reference: G. H. Elder, J. A. Tovey, and D. A Sheppard, Biochem. J., 215, 45-55 (1983).