Affinity-Based Strategies for
PROTEIN PURIFICATION Some innovative techniques are changing the way biochemists purify proteins.
T
he separation of proteins is almost as ancient an area of research as the discipline of biochemistry itself. The post-recombinant-DNA era is a promising time, in which the sourcing of proteins with the desired biological activity has become a more straightforward exercise (1). For example, sheep no longer must be killed to isolate insulin because it can be grown using recombinant E. coli cells. Kalyani Mondal At the same time, the post-genomic world has brought its own urgency Munishwar N. Gupta and requirements, as the contexts in which protein separations must be car- Indian Institute of Technology, ried out have increased (2). In the analytical context, electrophoretic Delhi techniques such as gel electrophoresis and CE dominate (3). In proteomics, Ipsita Roy 2D gel electrophoresis (with or without coupling to MS) is the most freNational Institute of quently used protein separation technique (2, 4 –6). In this article, we focus Pharmaceutical Education on the expanding range of tools for protein separation when purified proand Research (India) tein is required for specific applications. Two paradigm shifts have occurred in this area: using affinity to develop nonchromatographic strategies and then applying these strategies either early in the purification or as a stand-alone approach. This is in contrast to the old approach of using affinity chromatography in the last stage merely as a polishing step.
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All aspects of protein production must take into account the end application. However, the design of downstream processing (protein purification) protocols critically depends on it, because the level of purity required depends on the end application. Likewise, the effort required—the number of steps and the strategies used—depends on the desired level of purity (Figure 1; 7, 8). For example, enzymes used in detergents; in organic synthesis in nonaqueous media (9); in the textile, leather, paper and pulp, and food processing industries; and starch-degrading enzymes do not require a high degree of purity. In glucose analysis, some level of contaminating proteins in the glucose oxidase is acceptable as long as it does not interfere with detecting the glucose. A moderate amount of effort is therefore required to ensure purity. Therapeutic proteins such as insulin, urokinase, streptokinase, and interferons require the highest level of purity. Especially worrisome is the presence of endotoxins, nucleic acids, infectious agents, and degraded and/or aggregated product variants.
Protein purification Most industrial enzymes are extracellular and are secreted out of the cell. In the case of intracellular proteins, physical methods (e.g., osmotic shock, freezing, thawing), mechanical methods (e.g., ultrasonication, homogenization, bead mills), and chemical and enzymatic methods (treatment with solvents, detergents, and enzymes) can be used to break down the cells to obtain the crude protein (10). The choice of which to use depends upon the type of cell. One also has to be cautious in ensuring that the protein is not inactivated. For example, excessive ultrasonication may denature the protein. Initial purification consists primarily of separating the solid 3500
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phase (such as cell debris) from the liquid phase. Microfiltration uses membranes that retain particles in the diameter range 0.1–10 µm and are good enough to remove cell debris. Centrifugation in batch centrifuges (with capacities up to several liters) or continuous centrifuges is preferred if the broth is viscous. If the viscosity is too high, the broth can be pretreated by adding basic proteins or by hydrolysis with nucleases and mild heat and pH to precipitate nucleic acids. To aid filtration, celite can be added. Isolation of the proteins is dominated by nonchromatographic approaches, most often precipitation with salts, organic solvents, and polymers (10). Precipitation with salt (“salting-out”) is the preferred technique. The propensity of an anion to salt-out a protein is governed by the Hofmeister series: SCN – < ClO4– < NO3– < Br– < Cl – < CH3COO– < SO42– < PO43–. Monovalent cations are preferred. Because PO43– at neutral pH is in equilibrium with HPO42– and H2PO4– (the latter are less effective at salting-out), SO42– is preferred. Cost, solubility, and density considerations make (NH4)2SO4 the ideal candidate. Despite the wide acceptance of the Hofmeister series, the exact molecular description of how it operates has remained debatable. One widely held hypothesis is based on the ability of the anions to make (kosmotropy) or break (chaotropy) hydrogen bonds. However, recent experiments suggest that ions do not significantly affect the overall hydrogen bonding in bulk water (11). Vibrational sum-frequency spectroscopy was used to study an octadecylamine monolayer spread on salt solutions. The results indicate that dispersion forces play an important role and that the Hofmeister series conforms to the ability of an anion to penetrate the alkyl-chain monolayer. Thus, SO 42–, which penetrates much less than SCN –, would not penetrate the hydrophobic interior of a protein to disrupt its native structure (11). One of the clearest explanations was provided by Grover and Ryall, who examined salting-out in the context of urolithiasis (12). Among water-miscible organic solvents, acetone and ethyl alcohol are the favorite choices. The temperature must be kept low to avoid denaturation of the proteins. Mechanistically, organic solvents reduce the proteins’ solubility by decreasing the medium’s dielectric constant (12). The stability of proteins’ native structures in water– cosolvent mixtures has been examined in the context of enzyme catalysis in “good and bad solvents” (13). Albumin purification from blood is the most important example of this approach. Poly(ethylene glycol) (PEG), dextran, and polyacrylates are important examples of water-soluble polymers used for precipitation. More work has been carried out with PEG than with other polymers (14). Unlike organic solvents, PEG does not denature proteins even if it is present at a high concentration. Shorter precipitation times and precipitates that are amenable to centrifugation are two reasons PEG is superior to salt and organic solvents as a precipitating agent. In addition, judicious adjustment of the pH and/or the temperature of the solution can sometimes result in a good separation. Isoelectric precipitation occurs at the isoelectric point of the target protein. For example, a thermostable protein such as Rhodothermus marinus xylanase can be cloned in a host organism
such as E. coli. If the cell extract is incubated at 65 C, the xylanase can be recovered in solution while the other proteins are precipitated (7). In the de Marco et al. approach, recombinant-DNA technology is used to obtain the protein as a fusion protein with a thermostable tag (15). Heat incubation precipitates the host E. coli proteins but not the fusion protein. The use of other types of tags can also lead to some interesting possibilities. When an elastin-like polypeptide tag is attached, the fusion protein can be made insoluble at a specific temperature (Figure 2; 16). Thus, precipitation is a very versatile tool.
Cell lysate Protease digestion (optional)
ELP transition, then centrifugation Resolubilized pellet Supernatant (discarded)
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ELP
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FIGURE 2. Purification of elastin-like polypeptide (ELP)-tagged proteins by precipitation.
Size-based separations
The target fusion protein is tagged with thermally responsive ELP. When the temperature is raised, the fusion protein is
Gel filtration is a convenient selectively precipitated. The precipitate resolubilizes at a lower temperature, yielding purified ELP–target-protein. If required, way of separating molecules on the ELP tag can be removed proteolytically and the target protein recovered after one thermal cycle. (Adapted from Ref. 16.) the basis of size in a chromatographic mode. A variety of gel-filtration media with different frac- chromatography). One recent interesting approach is to use tionation ranges, chemical stabilities, and flow properties are com- two or three membrane composites (19). The highly selective mercially available (17). Bigger protein molecules do not enter and complete separation of 2 proteins with a molecular-weight the pores of the chosen beads, whereas smaller molecules can ratio of 1.03 was obtained this way. freely enter the beads and are retained to a much greater extent. The analogy of a sieve is often used to explain this process. How- Liquid/liquid extraction ever, it is not entirely correct because gel-filtration media attempt This well-established technique exploits partition of the desired to retain the smaller molecules. For this reason, gel filtration can protein into one of the phases in polymer/polymer (e.g., polyalso be used to remove salts and other low-molecular-weight im- mer/dextran) or polymer/salt (e.g., PEG/salt) two-phase syspurities from a protein solution. Gel filtration suffers from the dis- tems. Its virtues are that preclarification is not necessary and that advantages of clogging if the feed solution is not extremely clear it is easily scalable. The phases have high water content (generaland the difficulty of scaling up to large volumes. Membrane-based ly >90%), so the milieu is not harsh for biological structures. separations are preferred at this stage—gel filtration is generally More often than not, the selectivity of the process is not very carried out at a later stage. high. Hence, in its original form, the aqueous two-phase system Ultrafiltration membranes retain particles in the range (ATPS) shows great promise as an isolation step. It does away 0.01– 0.1 µm and operate in the pressure range 0.5–10 bar to with the need for centrifugation or filtration and concentrates fractionate proteins. These membranes can be obtained in tubu- the protein in the feed—any purification that occurs should be lar, plate-and-frame, spiral, and hollow formats. Earlier work was considered a bonus. characterized by high throughput and low resolution. ImproveWhen designing an ATPS, one must consider that higher-moments in this area are directed toward selectivity while maintain- lecular-weight PEGs require lower concentrations of the polymer ing high throughput. Another challenge is to reduce fouling and salt to form the two phases. However, as PEG becomes prefcaused by high-molecular-weight substances accumulating on erentially hydrated, the exclusion effect (precipitation) becomes the membrane’s surface. Various techniques have been tried to more pronounced with the higher-molecular-weight polymer. reduce fouling, such as rotating or vortex systems, high-frequen- Hence, the choice of the most suitable PEG vis-à-vis a target cy back-pulsing, cross-flow filtration, and high-performance tan- protein is best made by experimental optimization. gential-flow filtration (18). Small multivalent anions work best for phase formation. Unlike conventional bead chromatography, membrane-based Again, because these anions are also effective precipitating processes are not diffusion-limited. Thus, binding capacity does agents, potassium phosphate or citrate salts (in which proteins not depend upon flow rate. Higher capacities have been ob- are more soluble than in sulfate) are generally used. Changing tained by using stacks of conventional membranes (membrane the pH in an ATPS can cause charge effects that may alter proJ U N E 1 , 2 0 0 6 / A N A LY T I C A L C H E M I S T R Y
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Top phase (%, w/w)
tein partitioning and the nature and concentration of the mode, expanded-bed and perfusion chromatographies can anionic species (in the case of polyvalent salts). Optimization is be used to deal with unclarified feed (22). required in each case. Each ATPS can be viewed in terms of a phase diagram that The concept of affinity consists of a binodal curve that shows the composition of the two Traditionally, affinity chromatography has been used as a last polconstituents at which phase separation would occur (Figure 3). ishing step. In this technique, a ligand with affinity for the target ATPSs are formed at compositions above the binodal curve. Any protein is linked to a matrix, such as agarose beads (23). This two components mixed with concentrations that correspond to affinity capture step provides a level of selectivity that is generala point P will separate into two phases that correspond to the ly much higher than is possible with other approaches. The concentrations of A and B. A line bound protein can be eluted by such as APB is called a tie line. All changing the pH and/or the ionic X points on a tie line represent some strength of the operating buffer, but A Two-phase constituent composition in the two the elution process can be made region phases, but they differ in relative even more selective by a proteinP phase volumes and hence would specific ligand. yield different partition coefficients One major shift has been in B Y for a particular protein. defining what constitutes an affiniSingle-phase The selectivity of an ATPS ty ligand. In earlier years, the search region process is defined by the partition for an appropriate affinity ligand Bottom phase (%, w/w) coefficient K of a protein in two was limited to molecules that had a phases: K = [P]1/ [P]2, where [P]1 relationship with the protein in vivo. Coenzymes and competitive inand [P]2 are the concentrations of hibitors were thus the favorite the protein in the top and bottom FIGURE 3. Phase composition in an ATPS. choices. Textile dyes and chelated phases, respectively, at equilibrium. The contributions of various factors to K in a polymer/salt ATPS metal ions were found to be robust ligands with selectivity to incan be represented by ln K = ln K polymer + ln K salt + ln K ligand + dividual proteins because of a fortuitous combination of nonln Kothers. ATPSs such as ethylene oxide-propylene oxide ran- covalent interactions (i.e., ionic, hydrophobic, hydrogen bonds); dom copolymer/dextran, PEG/starch, PEG/polyacrylic acid, this approach was sometimes referred to as “pseudoaffinity poly(vinyl alcohol) (PVA)/dextran, and PEG/PVA have been chromatography” (17). With the advent of molecular modeling described (20). Fairly high selectivity has been reported in some and docking, dyes and other ligands were designed to more ATPSs. Many industrial enzymes, such as penicillin acylase and closely resemble substrate structures; these are called biomimetfumarase, have been isolated by ATPSs with purification factors ic ligands. Phage display and combinatorial peptide synthesis techniques of 5–25. Hence, in fortuitous circumstances, ATPSs may serve as more than just an isolation technique for proteins. Some out- enable the creation of peptide libraries. When coupled with highstanding examples of large-scale operations of ATPSs are throughput screening, such libraries allow the identification of a PEG/dextran for formate dehydrogenase from 45 kg Candida potential affinity ligand for a particular protein molecule. The boidinii, leucine dehydrogenase from Bacillus cereus from 70 L systematic evolution of ligands by exponential enrichment is a of cell homogenate, and interferon- from 40 L of mouse-cell similar approach that creates RNA and DNA oligonucleotide libraries (aptamers) and identifies a potential affinity ligand by supernatant (20). For most industrial applications of enzymes, the isolation screening (24). Thus, an affinity ligand may not have an in vivo strategies already discussed yield enzyme preparations of ade- relationship with the target protein. It is sufficient that the ligand quate purity. For other applications, additional purification is (irrespective of its chemical structure) binds to the target protein needed. At this point, the target protein is in a fairly concentrat- with a binding constant of 104–108 M–1. ed form and is relatively free of contamination. Some protein Currently, recombinant DNA technology enables the proproperties can be taken advantage of to effect a chromatograph- duction of the desired protein with a fusion affinity tag. One of ic separation (via gel filtration or ion-exchange or hydrophobic- the early popular tags was polyHis, which exploited the affinity interaction chromatographies) to further purify the sample (17). of histidines toward chelated metal ions (25). Today, quite a few The great variety of commercially available chromatographic commercially available options exist for obtaining the target promedia and the vast amount of “how-to” literature testify to the tein with tags such as cellulose binding-domain, chitin bindingpopularity of these techniques. However, these techniques are domain, and polyarginine (26). expensive; they can be difficult to scale up; and clogged columns Another major trend is the combination of affinity with can occur if the feed is not sufficiently clear. nonchromatographic approaches. Nonchromatographic separaSome high-throughput automated platforms have been de- tion methods such as ATPSs, membrane-based separations, and veloped, for example, AkTAxpress and Freedom EVO (21). Al- precipitation offer the convenience of handling large volumes though conventional ion-exchange/hydrophobic-interaction and are relatively inexpensive, although they lack the high selecchromatography of clarified feed is performed in packed-bed tivity of chromatographic methods. 3502
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in sequence. For example, -glucosidase can be purified by seOne approach, called affinity precipitation, combines the con- quential affinity precipitation with two different smart polymers venience of precipitation with the selectivity of bioaffinity. The (36). In the first step, the contaminant proteins are precipitated addition of a suitable, smart polymer–affinity-ligand conjugate to and removed; in the second step, -glucosidase is precipitated a crude broth allows the target protein to be captured. (“Smart” with chitosan. Similarly, two MLFTPP steps can be used in semeans that the ligand is reversibly soluble and insoluble in re- quence to separate and purify pectinase and cellulase from a bacsponse to a stimulus such as pH or temperature.) The complex terial source (35). An important and common design element in some of the can be precipitated by a suitable stimulus that leaves all the other contaminants in the solution. The precipitate can be separated work described is the exploitation of the often unexpected affinand the target protein dissociated and recovered. This process ity of smart polymers for the target protein. Obviously, the simdoes not require any of the costly equipment needed for affinity ple, straightforward application of polymers (without the need to chromatography. Scaling up is also easy. One unexpected but im- conjugate an affinity ligand) is a very desirable option—affinity mensely useful observation is that some smart polymers exhibit ligands tend to be expensive. Also, the price of conjugation is inherent affinity for a variety of useful enzymes. The affinities of often a significant percentage of the overall cost of the affinity a methyl methacrylate polymer, Eudragit S-100, for xylanase and matrix. Lastly, most affinity ligands tend to leach off the matrix of alginate for amylases, pectinases, and lipases have been exploited to precipitate enzymes. Other examples are the affiniThere is definitely a trend toward reducing ty precipitation of lysozyme, chitinases, the number of steps in protein-purification and chitin-binding lectins, all by chitosan, a pH-responsive polymer. procedures by incorporating affinity Applications of ATPSs in industry are rather limited because selectivity is not interactions. very high (hence purification is limited), the polymers are costly, and separating proteins from the PEG phase is difficult. The selectivity problem was solved by linking affinity ligands to during application. The presence of such unwanted chemicals PEG; this made the target protein selectively partition to the and biochemicals, even in trace amounts, causes problems in PEG phase. Cost has been addressed by replacing the PEG/dex- pharmaceutical and medical applications. However, the smart polymers have affinity for only a limited tran system with PEG/salt and PEG/starch systems. The separation problem can be solved by incorporation of a smart affini- range of proteins. At the same time, a variety of proteins tend to ty macroligand in the PEG phase and separation or precipitation bind to such polymers nonspecifically, thereby lowering the seof the ligand–protein complex with an appropriate stimulus. The lectivity of the process. Therefore, the selectivity of the polymer PEG can then be recovered and reused. Recently, this procedure can be modulated to either broaden or narrow the specificity. has been successfully used to purify amylases, xylanase, pullu- The selectivity of a smart polymer toward a population of proteins can be altered by microwave irradiation (37 ). In one case, lanase, and green fluorescent protein (27–29). Another, less well known, method is three-phase partitioning affinity precipitation with a microwave-treated polymer resulted (TPP). From undergraduate chemistry, we know that water-sol- in a higher-purity protein (38). Preliminary indications are that uble organic solvents separate out as a different phase in the pres- this result originates in the change in the surface hydrophobicity ence of salt (30). If a protein is present in a solution of ammoni- of the smart polymer (37 ). But for low selectivity, membrane-based separations are powum sulfate in water and tert-butanol is added, three phases result: the upper tert-butanol phase, the lower aqueous phase, and a erful approaches for protein separation. Polymers can be funcprecipitate of protein at the interface (31). Although the mech- tionalized with affinity ligands in the design of affinity-ligandanism of this TPP is far from clear, the technique matches the linked membranes. As early as 1987, affinity cross-flow filtration more well known two-phase affinity extraction in simplicity, scal- was successfully used with a variety of affinity ligands to purify different proteins (39). The application of stacked affinity memability, and ability to deal with unclarified feed. The use of smart polymer–affinity-ligand conjugates is espe- branes has enhanced both the selectivity and the capacity of the cially valuable in this case. Because smart polymers can be pre- binding process (40). Most of this work has been carried out cipitated at the interface by TPP (32), any target protein that is with dyes used as affinity ligands (41). Other interesting appresent will form a complex and float as an interfacial precipitate. plications are the separation of maltose binding protein on amyThis powerful separation method, called macroaffinity-ligand-fa- lose-linked cellulose membranes (42), purification of immunocilitated three-phase partitioning (MLFTPP), has already been globulins by protein-A-linked membranes (43), purification of successful in purifying -amylase (33), glucoamylase (34), pullu- penicillin acylase G on metal-linked membranes (44), purification of Tat protein (critical for HIV replication) on avidinlanase (34), xylanase (32), pectinase, and cellulase (35). Just as affinity chromatography can be used in a sequence of linked membranes (45), and fractionation of human plasma prodifferent affinity columns, these methods can also be performed teins by hydrophobic membrane chromatography (46).
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Conclusion Will affinity-based procedures, with their higher selectivity, replace other techniques? Not likely. Affinity ligands with relatively higher selectivity (e.g., monoclonal antibodies, biomimetic ligands, lectins) are costly. Chromatographic methods such as ion-exchange and hydrophobic-interaction chromatographies compete reasonably well with affinity-based separations, especially when high purity is not required. Also, in the case of pharmaceutical proteins or food processing enzymes, the risk always exists that affinity ligands will slowly dissociate from the affinity media. For this reason, the quest for ideal coupling chemistry continues, despite the many options. Nevertheless, a definite trend is toward reducing the number of steps in protein purification by incorporating affinity interactions. In a recent overview, Przybycien et al. evaluated alternatives to traditional packed-bed chromatography (47 ). Affinity precipitation and MLFTPP were described as being at the pre-commercial stage of industrial maturity, although precipitation was said to have reached high industrial maturity. Because both TPP and MLFTPP are essentially precipitation techniques, these nonchromatographic techniques may be the future of protein production. We acknowledge partial financial support provided by the various funding arms of the Government of India, namely the Department of Science and Technology and the Council of Scientific and Industrial Research (Extramural Division and Technology Mission on Oilseeds, Pulses and Maize). The financial support provided by the Indian Institute of Technology, Delhi, to KM in the form of a senior research fellowship is also gratefully acknowledged.
Kalyani Mondal, a graduate student at the Indian Institute of Technology, focuses her research on purification of proteins with smart polymers, microwave pretreatment of natural polysaccharides for enhanced biodegradability, biocatalyst design, and protein folding with smart polymers used as pseudochaperones. Ipsita Roy is on the biotechnology faculty at the National Institute of Pharmaceutical Education and Research (India). Her research interests include downstream processing of proteins, biocatalyst design, microwave-assisted enzymology, and aptamer technology. Munishwar N. Gupta is a professor and dean at the Indian Institute of Technology. His research interests include protein bioseparation, nonaqueous enzymology, and protein folding. Address correspondence to Gupta at Department of Chemistry, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi 110 016, India (
[email protected]).
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