CHAPTER FIVE
Use and Application of Hydrophobic Interaction Chromatography for Protein Purification Justin T. McCue1 Biogen Idec Corporation, 14 Cambridge Center, Cambridge, MA, USA 1 Corresponding author: e-mail address: [email protected]
Contents 1. Theory 1.1 Latest technology in HIC adsorbents 1.2 Advantages and disadvantages of using HIC 2. Equipment 3. Materials 3.1 Solutions & buffers 3.2 Preparation 4. Protocol 4.1 Preparation 4.2 Duration 5. Step 1 Column Equilibration 5.1 Overview 5.2 Duration 6. Step 2 Column Loading 6.1 Overview 6.2 Duration 7. Step 3 Product Elution 7.1 Overview 7.2 Duration 7.3 Gradient elution 7.4 Stepwise (isocratic) elution 8. Step 4 Adsorbent Regeneration and Sanitization 8.1 Overview 8.2 Duration References Source References
Methods in Enzymology, Volume 541 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-420119-4.00005-7
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2014 Elsevier Inc. All rights reserved.
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Abstract The objective of this section is to provide the reader with guidelines and background on the use and experimental application of Hydrophobic Interaction chromatography (HIC) for the purification of proteins. The section will give step by step instructions on how to use HIC in the laboratory to purify proteins. General guidelines and relevant background information is also provided.
1. THEORY Hydrophobic proteins will self-associate, or interact, when dissolved in an aqueous solution. This self-association forms the basis for a variety of biological interactions, such as protein folding, protein–substrate interactions, and transport of proteins across cellular membranes ( Janson and Ryde´n, 1997). Hydrophobic Interaction chromatography (HIC) is used in both analytical and preparatory scale protein purification applications. HIC exploits hydrophobic regions present in macromolecules that bind to hydrophobic ligands on chromatography adsorbents. The interaction occurs in an environment that favors hydrophobic interactions, such as an aqueous solution with a high salt concentration. By itself, water (a polar solvent) is a poor solvent for nonpolar molecules. Under such an environment, proteins will self-associate, or aggregate, in order to achieve a state of lowest thermodynamic energy. Prior to selfassociation, water molecules form highly ordered structures around each individual macromolecule (Fig. 5.1(a)). The self-association of nonpolar molecules (such as proteins) in the polar solvent is driven by a net increase in entropy of the environment. During the aggregation process, the overall surface area of hydrophobic sites of the protein exposed to the polar solvent is decreased, which results in a less structured (higher entropy) condition, which is the favored thermodynamic state. This same concept is responsible for the interaction (association) between hydrophobic ligands attached to an adsorbent and the proteins of interest (Fig. 5.1(b)). Association, or hydrophobic interaction, between the protein and the hydrophobic ligand is driven primarily by an increase in the overall entropy (compared with the condition when no interaction is occurring between the protein and the adsorbent). The polarity of the solvent can be controlled through the addition of salts or organic solvents, which can strengthen or weaken hydrophobic interactions between the HIC adsorbent and the protein. The influence of ions on
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Figure 5.1 Schematic diagram showing hydrophobic interactions between proteins in an aqueous solution (a), and between proteins and a hydrophobic ligand on an HIC adsorbent (b).
hydrophobic interaction follows the well-known Hofmeister series (Hofmeister, 1988). Anions that promote hydrophobic interaction are listed in decreasing strength of interaction, from left to right (Pa˚hlman et al., 1977): PO4 3 > SO4 2 > CH3 COO > Cl > Br > NO3 > CLO4 > I > SCN Ions that promote hydrophobic interactions are called lyotropes, while those that disrupt (weaken) hydrophobic interactions are called chaotropes. In the above series, phosphate ions promote the strongest hydrophobic interaction, while thiocyanate ions disrupt hydrophobic interactions. For cations, the Hofmeister series consists of the following (listed in order of decreasing lyotropic strength): NH4 þ > Rbþ > Kþ > Naþ > Csþ > Liþ > Mg2þ > Ca2þ > Ba2þ Two of the most common lyotropic salts used to promote hydrophobic interaction in aqueous solution are ammonium sulfate and sodium chloride. These salts are commonly employed when using HIC for protein purification. In addition to salts, organic solvents can also be used to alter the strength of hydrophobic interactions (Melander and Horvath, 1977; Fausnaugh and Regnier, 1986). Organic solvents commonly used to weaken or disrupt hydrophobic interactions include glycols, acetonitrile, and alcohols. The
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organic solvents alter the polarity of the mobile phase, thereby weakening potential interactions that may occur. They may be added to the solution during the elution process in order to disrupt hydrophobic interactions and elute the strongly bound protein of interest. Protein hydrophobicity is a complex function of several properties, which include the amino acid sequence, as well as protein tertiary and quaternary structure in a given solution (Ben-Naim, 1980; Tanford, 1980). Hydrophobicity scales have been created for particular amino acids, which are based upon the solubility in water and organic solvents (Tanford, 1962; Zimmerman et al., 1968; Nozaki and Tanford, 1971; Jones, 1975). Empirical hydrophobic scales for proteins have also been created (Chotia, 1976; Manavalan and Ponnuswamy, 1978; Wertz and Scheraga, 1978; Krigbaum and Komoriya, 1979; Rose et al., 1985) which are based upon the fraction of amino acids exposed on the protein surface, as well as the degree of amino acid hydrophobicity. The ability to predict the hydrophobicity of complex proteins has been only semiquantitative to date, and experiments are usually required to accurately understand protein hydrophobicity in a given aqueous solution.
1.1. Latest technology in HIC adsorbents HIC adsorbents consist of a base matrix that is coupled to a hydrophobic ligand. The base matrix, which typically consists of porous beads with diameters ranging from 5200 mm, provides a high surface area for ligand attachment and protein binding. Common base matrices include agarose, methacrylate, polystyrene–divinylbenzene, and silica (Table 5.1). For analytical applications, the bead size of the adsorbent is in the lower range (520mm). Small beads are used in order to maximize resolution when performing analytical separations. For preparatory scale applications, larger bead sizes are usually required (20mm). Larger bead sizes are required for preparatory scale columns due to pressure drop limitations associated with the column hardware. HIC adsorbents containing hydrophobic ligands with various degrees of hydrophobicity are available. The ligands consist of alkyl or aryl chains. As a general rule, the strength of hydrophobic binding of the ligand will increase with the length of the organic chain. Several of the most common ligands include butyl, octyl, and phenyl, which are linked to the base bead support through several different coupling approaches (Ulbrich et al., 1964; Hjerte´n et al., 1974). Aromatic ligands, such as phenyl, can also interact with the
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Table 5.1 Properties of commercially available HIC adsorbents Base matrix Available ligand types Adsorbent manufacturers
Cross-linked agarose
1. Butyl
GE Healthcare
2. Octyl 3. Phenyl Polystyrene divinylbenzene
Phenyl
GE Healthcare Applied Biosystems
Methacrylate
1. Butyl
TosoHass
2. Ether
EM Industries
3. Phenyl 4. Hexyl Silica
1. Propyl
JT Baker
2. Diol
Synchrom
3. Pentyl
Supelco YMC
adsorbed compounds through so-called ‘p–p interactions,’ which can further strengthen the hydrophobic interaction (Porath and Larsson, 1978). The hydrophobic interaction strength of the ligand can also be influenced by the ligand loading (ligand density) on the base matrix. The strength of interaction can increase with higher ligand densities. In order to have reproducible performance, manufacturers of HIC adsorbents must often produce adsorbents with narrow ranges of ligand density to ensure consistent performance from lot to lot.
1.2. Advantages and disadvantages of using HIC HIC is most commonly employed when aggregated protein species need to be separated from a more desirable monomeric form. HIC often possesses superior selectivity for removal of aggregate species, compared to other forms of chromatography, including ion exchange and affinity (see Using ion exchange chromatography to purify a recombinantly expressed protein, Purification of His-tagged proteins, Affinity purification of a recombinant protein expressed as a fusion with the maltose-binding protein (MBP) tag, Purification of GST-tagged proteins, Protein Affinity Purification using
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Intein/Chitin Binding Protein Tags, Immunoaffinity purification of proteins and Strep-tagged protein purification). HIC may also provide superior selectivity for the removal of undesirable misfolded or variant forms of a protein. Use of HIC often requires the use of high salt concentrations to ensure sufficient hydrophobic interaction between the protein and the adsorbent. Buffers containing high concentrations of salt may be costly to produce or may be expensive to dispose of properly (depending on the existing environmental disposal requirements). In such cases, use of other forms of chromatography may be more desirable.
2. EQUIPMENT ¨ KTAexplorerTM, GE Healthcare, or Chromatography system (e.g., A BioCAD Vision Workstation, PerSeptive Biosystems) Chromatography columns UV Spectrophotometer Analytical balance Glassware (beakers, graduated cylinders) Pipettes Analytical weighing trays Magnetic stir plate Stir bars 0.2-mm filters
3. MATERIALS Sodium phosphate, monobasic (NaH2PO4H2O) Sodium phosphate, dibasic (Na2HPO4 7H2O) Sodium chloride (NaCl) Ammonium sulfate [(NH4)2SO4] Magnesium chloride (MgCl2) Sodium acetate (NaOAc3H2O) Acetic acid (glacial) Sodium hydroxide (NaOH) Ethanol Propylene glycol Ethylene glycol Guanidine HCl Deionized water
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3.1. Solutions & buffers Note: The precise buffer formulations are highly dependent on the protein to be purified and the HIC adsorbent being used and must be determined empirically. Therefore, only general suggestions or options for buffers are given.
3.2. Preparation Protein Load adjustment solution options
Option Option Option Option
1: 50-mM Phosphate þ 3-M Ammonium Sulfate, pH 7 2: 50-mM Phosphate þ 3-M Sodium Chloride, pH 7 3: 50-mM Acetate þ 3-M Ammonium Sulfate, pH 5 4: 50-mM Acetate þ 3-M Sodium Chloride, pH 5 50-mM Phosphate buffer, pH 7 Component
Amount
NaH2PO4 H2O
2.9g
Na2HPO4 7H2O
7.7g
Dissolve in a total volume of 1 l of purified water. Pass through a 0.2-mm filter to sterilize
50-mM Acetate buffer, pH 5 Component
Amount
NaOAc3H2O
4.36 g
Glacial acetic acid
1.1g
Dissolve in a total volume of 1 l of purified water. Pass through a 0.2-mm filter to sterilize
Steps 1 and 2 Column equilibration and column wash buffer
The buffer used to equilibrate and wash the HIC column should contain a salt concentration and pH at a similar level as the load-adjusted protein solution Step 3 Column elution buffer
Option 1: 50-mM Phosphate þ 0–1.0 M Ammonium Sulfate, pH 7 Option 2: 50-mM Phosphate þ 0–1.0 M Sodium Chloride, pH 7 Option 3: 50-mM Acetate þ 0–1.0 M Ammonium Sulfate, pH 5 Option 4: 50-mM Acetate þ 0–1.0 M Sodium Chloride, pH 5
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Step 4 Column regeneration and sanitization buffers
0.1–1.0-M NaOH 6-M Guanidine HCl 20–100% Ethanol
Column storage buffers
20% Ethanol 0.1-M NaOH
4. PROTOCOL 4.1. Preparation Determine the salt concentration needed for the protein-containing solution. It must be high enough that the protein binds to the HIC adsorbent, but not so high that the protein precipitates out of solution. The pH of the protein-containing solution should also be varied so that both the protein and the adsorbent are stable.
4.2. Duration Preparation
About 2 days
Protocol
About 1 day
Comments Prior to column loading, the salt concentration of the protein mixture (which will be purified using the HIC adsorbent) must be increased to a level in which the target protein binds to the adsorbent using a high salt buffer. Proteins may precipitate in high salt solutions, so the compound solubility in the salt solution should be evaluated prior to the HIC chromatography experiments being initiated. The salt concentration of the protein load should be adjusted to a range in which the protein is known to be soluble. A buffer pH should be chosen in which the protein and the adsorbent are stable (e.g., avoid using the extreme ends of the pH scale). As a general rule of thumb, a pH range of 5–7 can be chosen as an initial starting range for the chromatography operation. During the protein load adjustment step, the salt
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Figure 5.2 Flowchart of the complete protocol, including preparation.
concentration may range from 0.52.0M, and will be increased high enough to ensure that the protein binds effectively to the adsorbent, but low enough such that protein precipitation does not occur. The salt concentration required to bind the protein to the adsorbent will depend greatly on the choice of salt, as described in the Theory section. Selection of the appropriate salt concentration in which the protein binds to the adsorbent will require experimental screening work and will vary significantly from protein to protein in most cases. See Fig. 5.2 for the flowchart of the complete protocol.
5. STEP 1 COLUMN EQUILIBRATION 5.1. Overview The column will be equilibrated with a buffer of similar composition (salt concentration and pH) to the buffer the protein is in.
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5.2. Duration Approximately 2 h 1.1 Wash the column with 3–5 column volumes of equilibration buffer. The flow rate should be between 50 and 300cmh1. Comments Prior to loading the protein feed, the column should first be equilibrated in a high salt buffer solution that possesses a similar composition (salt concentration) and pH as the feed solution to ensure that the protein will bind tightly to the adsorbent. This step is referred to as the equilibration step. The operating flow rate should be chosen in a range in which the HIC adsorbent is known to be stable. Flow-rate ranges are often provided by the HIC adsorbent manufacturer, so the user should consult the available guidelines prior to operation. As a general rule, HIC adsorbents (at laboratory scale) are operated at a superficial velocity of 50–300 cmh1.
6. STEP 2 COLUMN LOADING 6.1. Overview The protein-containing solution is loaded onto the column, allowing the protein to bind to the HIC adsorbent. Nonspecifically bound constituents are washed from the column.
6.2. Duration Approximately 2 h 2.1 Load the protein-containing solution onto the column. 2.2 Wash the column with 3–10 column volumes of equilibration buffer over a period of 1–2 h. Comments Following the equilibration step, the adjusted feed (which contains the protein of interest) is loaded onto the HIC column. During the load step, the protein binds to the adsorbent. After the protein-containing solution is loaded onto the column, the column can be washed with the equilibration buffer prior to product elution. Approximately 3–10 column volumes can be used for the wash buffer. Additional wash steps may be implemented prior to the elution step to remove undesirable impurity species which are bound to the adsorbent. The wash steps may contain a salt concentration at an intermediate salt concentration which is less than the load step but greater than the elution step.
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7. STEP 3 PRODUCT ELUTION 7.1. Overview The protein is eluted from the column using a linear (decreasing) gradient of salt concentration, separating it from impurities or unwanted species.
7.2. Duration Approximately 3 h 3.1 Elute the column using a decreasing linear gradient of salt concentration (e.g., from 1 M to 0 M salt) over 10 column volumes of buffer. 3.2 Collect fractions and analyze the gradient profile for the presence of protein (e.g., by measuring absorbance at 254nm). Comments After performing the wash step(s), the desired protein must be eluted and then collected in the column effluent. In many cases, the elution process is used to separate, or resolve, unwanted species from the desired protein. The unwanted species may bind less tightly to the adsorbent and will be eluted prior to the product. In other cases, undesirable species bind more tightly to the adsorbent and will remain bound after the product is eluted. This is usually the case when HIC is used to separate protein aggregate species, which bind more tightly to the adsorbent than the desired protein monomer species. During the elution step, a portion (or fraction) of the eluate may contain highly purified product, while fractions before and after contain higher levels of undesirable impurities. A schematic of an elution process (during a gradient elution) is shown in Fig. 5.3. Figure 5.3 illustrates that the column effluent collected during the elution step may need to be fractionated in order to achieve acceptable product purity when using HIC. The elution process can be done using either a stepwise (isocratic) or a gradient approach. The four most common methods (listed from most common to least common) used to elute the bound protein include the following: i. Decrease in the salt concentration (relative to the binding conditions). A decrease in the salt concentration will decrease the strength of hydrophobic interactions between the protein and the ligand, and the protein will be desorbed and eluted from the column. ii. Addition of organic solvents. Addition of an organic solvent (such as ethylene or propylene glycol) changes the solvent polarity, which disrupts the hydrophobic interaction.
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Figure 5.3 Schematic chromatogram showing a gradient elution of a protein mixture using hydrophobic interaction chromatography. In the diagram, the salt concentration is linearly decreased (from high salt to low salt), which results in elution of both impurities and the target protein.
iii. Increase in the salt concentration (using a chaotropic salt). Addition of a chaotropic salt will disrupt the hydrophobic interaction. iv. Detergent addition. Detergents are used as protein displacers, and have been used mainly for the purification of membrane proteins when using HIC ( Janson and Ryde´n, 1997, see also Explanatory Chapter: Choosing the right detergent). This most common approach used to elute proteins from HIC adsorbents is by lowering the salt concentration during the elution step. This should be the first method that is attempted when using HIC for purification of a new protein compound. The other approaches described above have the disadvantage that an additional component (such as a chaotropic salt or an organic solvent) needs to be added, which may impact protein stability. However, such agents may be required in order to effectively elute a strongly bound protein species from the adsorbent. Each protein must be evaluated case by case to determine which elution method is appropriate. The HIC adsorbent used in the purification may also influence which elution method is effective.
7.3. Gradient elution Gradient elutions are an extremely effective method useful for screening different HIC adsorbents in protein purification. During the gradient elution
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process, the salt concentration is decreased gradually (in a linear fashion) from a high salt concentration to a low salt concentration over a defined volume. During the initial screening of a bound compound on an adsorbent, the salt concentration may be decreased to as low as 0 mM to determine the salt concentration when the product elutes. As a starting point, a typical gradient elution process is performed over ten (10) column volumes, during which fractions are collected and evaluated for product purity. The gradient in salt concentration may be decreased (performed over a larger volume) in order to improve protein resolution (Yamamoto et al., 1988). In the event that the protein remains bound to the adsorbent following the gradient elution process, this may indicate that either a weaker lyotropic salt should be selected to bind the protein to the adsorbent, or that a stronger elution condition is required to elute the protein. Stronger elution solutions may include the use of an organic solvent, including propylene glycol, ethylene glycol, acetonitrile, or ethanol. Alternatively, an adsorbent with weaker hydrophobic binding strength may need to be selected to decrease the strength of hydrophobic interaction and achieve an acceptable product recovery.
7.4. Stepwise (isocratic) elution After identifying the appropriate adsorbent and salt concentration to effectively elute the protein of interest, an isocratic elution can be used if desired. An advantage of using isocratic elution is its simplicity – it requires a simple switch in the inlet buffer (from a high to a low salt concentration). Use of an isocratic elution is a preferable approach to simplify the equipment requirements, as gradient elution requires multiple pumps and additional process control to generate a linear change in the buffer salt concentration.
8. STEP 4 ADSORBENT REGENERATION AND SANITIZATION 8.1. Overview The HIC adsorbent will be washed, regenerated, and placed into storage buffer for reuse.
8.2. Duration 3–6 h
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4.1 Wash the column using 3–5 column volumes of regeneration buffer (e.g., 6-M guanidine hydrochloride, or 20–100% ethanol or methanol) over a period of 1–2 h. 4.2 Wash the column using 3–5 column volumes of sanitization buffer (e.g., 1-M NaOH) over a period of 1–2 h. 4.3 Wash the column using 3–5 column volumes of storage buffer (e.g., 20% ethanol or 0.1M NaOH) over a period of 1–2h. Comments: HIC adsorbents are reusable for multiple cycles and have a relatively long lifetime before having to be replaced. However, adsorbents must be cleaned and regenerated between uses in order to ensure reproducible performance over many cycles. The adsorbent manufacturers provide regeneration procedures for the adsorbents, which can be consulted prior to use. In general, the cleaning procedures depend upon the stability of the base matrix and the hydrophobic ligand. For strongly bound proteins, 6-M guanidine hydrochloride is often recommended. If detergents have been used during the process, ethanol or methanol can be used as part of the regeneration procedure (GE Healthcare, 2006). For sanitization, a caustic solution (1.0-M NaOH) can be used for most of the adsorbents (with the exception of silica). The adsorbent manufacturer should also provide information on the appropriate storage conditions. In a typical process, the HIC column is exposed to subsequent regeneration and sanitization solutions for 1–2 h for each step, using 3–5 column volumes. Following the regeneration and sanitization steps, a storage solution should be selected that prevents microbial growth, but does not impact ligand or base matrix stability. For the storage step, 3–5 column volumes of storage solution should be passed through the column to ensure that the column is properly equilibrated prior to storage.
REFERENCES Referenced Literature Ben-Naim, A. (1980). Hydrophobic Interactions. New York: Plenum Press. Chotia, C. (1976). Surface of monomelic proteins. Journal of Molecular Biology, 105, 1–12. Fausnaugh, J. L., & Regnier, F. E. (1986). Solute and mobil phase contributions to retention in hydrophobic interaction chromatography of proteins. Journal of Chromatography, 359, 131–146. GE Healthcare (2006). Data File No. 18-1127-63 AC. Hjerte´n, S., Rosengren, J., & Pa˚hlman, S. (1974). Hydrophobic interaction chromatography. Journal of Chromatography, 101, 281–288. Hofmeister, F. (1988). On regularities in the albumin precipitation reactions with salts and their relationship to physiological behavior. Archiv for Experimentelle Pathologie und Pharmakologie, 24, 247–260.
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Janson, J.-C., & Ryde´n, L. (Eds.), (1997). Protein Purification: Principles, High-Resolution Methods, and Applications (2nd edn., p. 284). Weinheim: Wiley-VCH. Jones, D. D. (1975). Amino acid properties and side-chain orientation in proteins. Journal of Theoretical Biology, 50, 167–183. Krigbaum, W. R., & Komoriya, A. (1979). Local interactions as a structure determinant for protein molecules. Biochimica et Biophysica Acta, 576, 204–248. Manavalan, P., & Ponnuswamy, P. K. (1978). Hydrophobic character of amino acid residues in globular proteins. Nature, 275, 673–674. Melander, W., & Horvath, C. (1977). Salt effects on hydrophobic interactions in precipitation and chromatography of proteins: An interpretation of the lyotropic series. Archives of Biochemistry and Biophysics, 183, 200–215. Nozaki, Y., & Tanford, C. (1971). The solubility of amino acids and two glycine peptides in aqueous ethanol and dioxane solutions. Establishment of a hydrophobicity scale. Journal of Biological Chemistry, 246, 2211–2217. Pa˚hlman, S., Rosengren, J., & Hjerten, S. (1977). Hydrophobic interaction chromatography on uncharged sepharose derivatives. Journal of Chromatography, 131, 99–108. Porath, J., & Larsson, B. (1978). Charge-transfer and water-mediated chromatography. I: Electron-acceptor ligands on cross-linked dextran Journal of Chromatography, 155, 47–68. Rose, G. D., Geselowitz, A. R., Lesser, G. J., Lee, R. H., & Zehfus, M. H. (1985). Hydrophobicity of amino acid residues in globular proteins. Science, 229, 834–838. Tanford, C. (1962). Contribution of hydrophobic interactions to the stability of the globular conformation of proteins. Journal of American Chemical Society, 84, 4240–4247. Tanford, C. (1980). The Hydrophobic Effect (2nd edn.). New York: Wiley. Ulbrich, V., Makes, J., & Jurecek, M. (1964). Identification of giycidyl ethers. Bis(phenyl-) and bis (a-naphthylurethans) of glycerol a-alkyl (aryl)ethers. Collection of Czechoslovak Chemical Communications, 29, 1466–1475. Wertz, D. H., & Scheraga, H. A. (1978). Influence of water on protein structure. Macromolecules, 11, 9–15. Yamamoto, S., Nakanishi, K., & Matsuno, R. (1988). Ion-Exchange Chromatography of Proteins. New York: Mercel Dekkar. Zimmerman, J. M., Eliezer, N., & Simha, R. (1968). The characterization of amino acid sequences in proteins by statistical methods. Journal of Theoretical Biology, 21(2), 170–201.
SOURCE REFERENCES Burgess, R., & Deutshcer, M. (Eds.), (2009). Methods in Enzymology, Guide to Protein Purification (2nd edn, pp. 405–413). New York: Elsevier, vol. 463. Hjerte´n, S., Yao, K., Eriksson, K.-O., & Johansson, B. (1986). Gradient and isocratic high performance hydrophobic interaction chromatography of proteins on agarose columns. Journal of Chromatography, 359, 99–109.
Referenced Protocols in Methods Navigator Using ion exchange chromatography to purify a recombinantly expressed protein. Purification of His-tagged proteins. Affinity purification of a recombinant protein expressed as a fusion with the maltose-binding protein (MBP) tag. Purification of GST-tagged proteins. Protein Affinity Purification using Intein/Chitin Binding Protein Tags. Immunoaffinity purification of proteins. Strep-tagged protein purification. Explanatory Chapter: Choosing the right detergent.
Use and Application of Hydrophobic Interaction Chromatography for Protein Purification Justin T. McCue1 Biogen Idec Corporation, 14 Cambridge Center, Cambridge, MA, USA 1 Corresponding author: e-mail address: [email protected]
Contents 1. Theory 1.1 Latest technology in HIC adsorbents 1.2 Advantages and disadvantages of using HIC 2. Equipment 3. Materials 3.1 Solutions & buffers 3.2 Preparation 4. Protocol 4.1 Preparation 4.2 Duration 5. Step 1 Column Equilibration 5.1 Overview 5.2 Duration 6. Step 2 Column Loading 6.1 Overview 6.2 Duration 7. Step 3 Product Elution 7.1 Overview 7.2 Duration 7.3 Gradient elution 7.4 Stepwise (isocratic) elution 8. Step 4 Adsorbent Regeneration and Sanitization 8.1 Overview 8.2 Duration References Source References
Methods in Enzymology, Volume 541 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-420119-4.00005-7
52 54 55 56 56 57 57 58 58 58 59 59 60 60 60 60 61 61 61 62 63 63 63 63 64 65
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2014 Elsevier Inc. All rights reserved.
51
52
Justin T. McCue
Abstract The objective of this section is to provide the reader with guidelines and background on the use and experimental application of Hydrophobic Interaction chromatography (HIC) for the purification of proteins. The section will give step by step instructions on how to use HIC in the laboratory to purify proteins. General guidelines and relevant background information is also provided.
1. THEORY Hydrophobic proteins will self-associate, or interact, when dissolved in an aqueous solution. This self-association forms the basis for a variety of biological interactions, such as protein folding, protein–substrate interactions, and transport of proteins across cellular membranes ( Janson and Ryde´n, 1997). Hydrophobic Interaction chromatography (HIC) is used in both analytical and preparatory scale protein purification applications. HIC exploits hydrophobic regions present in macromolecules that bind to hydrophobic ligands on chromatography adsorbents. The interaction occurs in an environment that favors hydrophobic interactions, such as an aqueous solution with a high salt concentration. By itself, water (a polar solvent) is a poor solvent for nonpolar molecules. Under such an environment, proteins will self-associate, or aggregate, in order to achieve a state of lowest thermodynamic energy. Prior to selfassociation, water molecules form highly ordered structures around each individual macromolecule (Fig. 5.1(a)). The self-association of nonpolar molecules (such as proteins) in the polar solvent is driven by a net increase in entropy of the environment. During the aggregation process, the overall surface area of hydrophobic sites of the protein exposed to the polar solvent is decreased, which results in a less structured (higher entropy) condition, which is the favored thermodynamic state. This same concept is responsible for the interaction (association) between hydrophobic ligands attached to an adsorbent and the proteins of interest (Fig. 5.1(b)). Association, or hydrophobic interaction, between the protein and the hydrophobic ligand is driven primarily by an increase in the overall entropy (compared with the condition when no interaction is occurring between the protein and the adsorbent). The polarity of the solvent can be controlled through the addition of salts or organic solvents, which can strengthen or weaken hydrophobic interactions between the HIC adsorbent and the protein. The influence of ions on
Use and Application of Hydrophobic Interaction Chromatography for Protein Purification
53
Figure 5.1 Schematic diagram showing hydrophobic interactions between proteins in an aqueous solution (a), and between proteins and a hydrophobic ligand on an HIC adsorbent (b).
hydrophobic interaction follows the well-known Hofmeister series (Hofmeister, 1988). Anions that promote hydrophobic interaction are listed in decreasing strength of interaction, from left to right (Pa˚hlman et al., 1977): PO4 3 > SO4 2 > CH3 COO > Cl > Br > NO3 > CLO4 > I > SCN Ions that promote hydrophobic interactions are called lyotropes, while those that disrupt (weaken) hydrophobic interactions are called chaotropes. In the above series, phosphate ions promote the strongest hydrophobic interaction, while thiocyanate ions disrupt hydrophobic interactions. For cations, the Hofmeister series consists of the following (listed in order of decreasing lyotropic strength): NH4 þ > Rbþ > Kþ > Naþ > Csþ > Liþ > Mg2þ > Ca2þ > Ba2þ Two of the most common lyotropic salts used to promote hydrophobic interaction in aqueous solution are ammonium sulfate and sodium chloride. These salts are commonly employed when using HIC for protein purification. In addition to salts, organic solvents can also be used to alter the strength of hydrophobic interactions (Melander and Horvath, 1977; Fausnaugh and Regnier, 1986). Organic solvents commonly used to weaken or disrupt hydrophobic interactions include glycols, acetonitrile, and alcohols. The
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organic solvents alter the polarity of the mobile phase, thereby weakening potential interactions that may occur. They may be added to the solution during the elution process in order to disrupt hydrophobic interactions and elute the strongly bound protein of interest. Protein hydrophobicity is a complex function of several properties, which include the amino acid sequence, as well as protein tertiary and quaternary structure in a given solution (Ben-Naim, 1980; Tanford, 1980). Hydrophobicity scales have been created for particular amino acids, which are based upon the solubility in water and organic solvents (Tanford, 1962; Zimmerman et al., 1968; Nozaki and Tanford, 1971; Jones, 1975). Empirical hydrophobic scales for proteins have also been created (Chotia, 1976; Manavalan and Ponnuswamy, 1978; Wertz and Scheraga, 1978; Krigbaum and Komoriya, 1979; Rose et al., 1985) which are based upon the fraction of amino acids exposed on the protein surface, as well as the degree of amino acid hydrophobicity. The ability to predict the hydrophobicity of complex proteins has been only semiquantitative to date, and experiments are usually required to accurately understand protein hydrophobicity in a given aqueous solution.
1.1. Latest technology in HIC adsorbents HIC adsorbents consist of a base matrix that is coupled to a hydrophobic ligand. The base matrix, which typically consists of porous beads with diameters ranging from 5200 mm, provides a high surface area for ligand attachment and protein binding. Common base matrices include agarose, methacrylate, polystyrene–divinylbenzene, and silica (Table 5.1). For analytical applications, the bead size of the adsorbent is in the lower range (520mm). Small beads are used in order to maximize resolution when performing analytical separations. For preparatory scale applications, larger bead sizes are usually required (20mm). Larger bead sizes are required for preparatory scale columns due to pressure drop limitations associated with the column hardware. HIC adsorbents containing hydrophobic ligands with various degrees of hydrophobicity are available. The ligands consist of alkyl or aryl chains. As a general rule, the strength of hydrophobic binding of the ligand will increase with the length of the organic chain. Several of the most common ligands include butyl, octyl, and phenyl, which are linked to the base bead support through several different coupling approaches (Ulbrich et al., 1964; Hjerte´n et al., 1974). Aromatic ligands, such as phenyl, can also interact with the
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Table 5.1 Properties of commercially available HIC adsorbents Base matrix Available ligand types Adsorbent manufacturers
Cross-linked agarose
1. Butyl
GE Healthcare
2. Octyl 3. Phenyl Polystyrene divinylbenzene
Phenyl
GE Healthcare Applied Biosystems
Methacrylate
1. Butyl
TosoHass
2. Ether
EM Industries
3. Phenyl 4. Hexyl Silica
1. Propyl
JT Baker
2. Diol
Synchrom
3. Pentyl
Supelco YMC
adsorbed compounds through so-called ‘p–p interactions,’ which can further strengthen the hydrophobic interaction (Porath and Larsson, 1978). The hydrophobic interaction strength of the ligand can also be influenced by the ligand loading (ligand density) on the base matrix. The strength of interaction can increase with higher ligand densities. In order to have reproducible performance, manufacturers of HIC adsorbents must often produce adsorbents with narrow ranges of ligand density to ensure consistent performance from lot to lot.
1.2. Advantages and disadvantages of using HIC HIC is most commonly employed when aggregated protein species need to be separated from a more desirable monomeric form. HIC often possesses superior selectivity for removal of aggregate species, compared to other forms of chromatography, including ion exchange and affinity (see Using ion exchange chromatography to purify a recombinantly expressed protein, Purification of His-tagged proteins, Affinity purification of a recombinant protein expressed as a fusion with the maltose-binding protein (MBP) tag, Purification of GST-tagged proteins, Protein Affinity Purification using
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Intein/Chitin Binding Protein Tags, Immunoaffinity purification of proteins and Strep-tagged protein purification). HIC may also provide superior selectivity for the removal of undesirable misfolded or variant forms of a protein. Use of HIC often requires the use of high salt concentrations to ensure sufficient hydrophobic interaction between the protein and the adsorbent. Buffers containing high concentrations of salt may be costly to produce or may be expensive to dispose of properly (depending on the existing environmental disposal requirements). In such cases, use of other forms of chromatography may be more desirable.
2. EQUIPMENT ¨ KTAexplorerTM, GE Healthcare, or Chromatography system (e.g., A BioCAD Vision Workstation, PerSeptive Biosystems) Chromatography columns UV Spectrophotometer Analytical balance Glassware (beakers, graduated cylinders) Pipettes Analytical weighing trays Magnetic stir plate Stir bars 0.2-mm filters
3. MATERIALS Sodium phosphate, monobasic (NaH2PO4H2O) Sodium phosphate, dibasic (Na2HPO4 7H2O) Sodium chloride (NaCl) Ammonium sulfate [(NH4)2SO4] Magnesium chloride (MgCl2) Sodium acetate (NaOAc3H2O) Acetic acid (glacial) Sodium hydroxide (NaOH) Ethanol Propylene glycol Ethylene glycol Guanidine HCl Deionized water
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3.1. Solutions & buffers Note: The precise buffer formulations are highly dependent on the protein to be purified and the HIC adsorbent being used and must be determined empirically. Therefore, only general suggestions or options for buffers are given.
3.2. Preparation Protein Load adjustment solution options
Option Option Option Option
1: 50-mM Phosphate þ 3-M Ammonium Sulfate, pH 7 2: 50-mM Phosphate þ 3-M Sodium Chloride, pH 7 3: 50-mM Acetate þ 3-M Ammonium Sulfate, pH 5 4: 50-mM Acetate þ 3-M Sodium Chloride, pH 5 50-mM Phosphate buffer, pH 7 Component
Amount
NaH2PO4 H2O
2.9g
Na2HPO4 7H2O
7.7g
Dissolve in a total volume of 1 l of purified water. Pass through a 0.2-mm filter to sterilize
50-mM Acetate buffer, pH 5 Component
Amount
NaOAc3H2O
4.36 g
Glacial acetic acid
1.1g
Dissolve in a total volume of 1 l of purified water. Pass through a 0.2-mm filter to sterilize
Steps 1 and 2 Column equilibration and column wash buffer
The buffer used to equilibrate and wash the HIC column should contain a salt concentration and pH at a similar level as the load-adjusted protein solution Step 3 Column elution buffer
Option 1: 50-mM Phosphate þ 0–1.0 M Ammonium Sulfate, pH 7 Option 2: 50-mM Phosphate þ 0–1.0 M Sodium Chloride, pH 7 Option 3: 50-mM Acetate þ 0–1.0 M Ammonium Sulfate, pH 5 Option 4: 50-mM Acetate þ 0–1.0 M Sodium Chloride, pH 5
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Step 4 Column regeneration and sanitization buffers
0.1–1.0-M NaOH 6-M Guanidine HCl 20–100% Ethanol
Column storage buffers
20% Ethanol 0.1-M NaOH
4. PROTOCOL 4.1. Preparation Determine the salt concentration needed for the protein-containing solution. It must be high enough that the protein binds to the HIC adsorbent, but not so high that the protein precipitates out of solution. The pH of the protein-containing solution should also be varied so that both the protein and the adsorbent are stable.
4.2. Duration Preparation
About 2 days
Protocol
About 1 day
Comments Prior to column loading, the salt concentration of the protein mixture (which will be purified using the HIC adsorbent) must be increased to a level in which the target protein binds to the adsorbent using a high salt buffer. Proteins may precipitate in high salt solutions, so the compound solubility in the salt solution should be evaluated prior to the HIC chromatography experiments being initiated. The salt concentration of the protein load should be adjusted to a range in which the protein is known to be soluble. A buffer pH should be chosen in which the protein and the adsorbent are stable (e.g., avoid using the extreme ends of the pH scale). As a general rule of thumb, a pH range of 5–7 can be chosen as an initial starting range for the chromatography operation. During the protein load adjustment step, the salt
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Figure 5.2 Flowchart of the complete protocol, including preparation.
concentration may range from 0.52.0M, and will be increased high enough to ensure that the protein binds effectively to the adsorbent, but low enough such that protein precipitation does not occur. The salt concentration required to bind the protein to the adsorbent will depend greatly on the choice of salt, as described in the Theory section. Selection of the appropriate salt concentration in which the protein binds to the adsorbent will require experimental screening work and will vary significantly from protein to protein in most cases. See Fig. 5.2 for the flowchart of the complete protocol.
5. STEP 1 COLUMN EQUILIBRATION 5.1. Overview The column will be equilibrated with a buffer of similar composition (salt concentration and pH) to the buffer the protein is in.
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5.2. Duration Approximately 2 h 1.1 Wash the column with 3–5 column volumes of equilibration buffer. The flow rate should be between 50 and 300cmh1. Comments Prior to loading the protein feed, the column should first be equilibrated in a high salt buffer solution that possesses a similar composition (salt concentration) and pH as the feed solution to ensure that the protein will bind tightly to the adsorbent. This step is referred to as the equilibration step. The operating flow rate should be chosen in a range in which the HIC adsorbent is known to be stable. Flow-rate ranges are often provided by the HIC adsorbent manufacturer, so the user should consult the available guidelines prior to operation. As a general rule, HIC adsorbents (at laboratory scale) are operated at a superficial velocity of 50–300 cmh1.
6. STEP 2 COLUMN LOADING 6.1. Overview The protein-containing solution is loaded onto the column, allowing the protein to bind to the HIC adsorbent. Nonspecifically bound constituents are washed from the column.
6.2. Duration Approximately 2 h 2.1 Load the protein-containing solution onto the column. 2.2 Wash the column with 3–10 column volumes of equilibration buffer over a period of 1–2 h. Comments Following the equilibration step, the adjusted feed (which contains the protein of interest) is loaded onto the HIC column. During the load step, the protein binds to the adsorbent. After the protein-containing solution is loaded onto the column, the column can be washed with the equilibration buffer prior to product elution. Approximately 3–10 column volumes can be used for the wash buffer. Additional wash steps may be implemented prior to the elution step to remove undesirable impurity species which are bound to the adsorbent. The wash steps may contain a salt concentration at an intermediate salt concentration which is less than the load step but greater than the elution step.
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7. STEP 3 PRODUCT ELUTION 7.1. Overview The protein is eluted from the column using a linear (decreasing) gradient of salt concentration, separating it from impurities or unwanted species.
7.2. Duration Approximately 3 h 3.1 Elute the column using a decreasing linear gradient of salt concentration (e.g., from 1 M to 0 M salt) over 10 column volumes of buffer. 3.2 Collect fractions and analyze the gradient profile for the presence of protein (e.g., by measuring absorbance at 254nm). Comments After performing the wash step(s), the desired protein must be eluted and then collected in the column effluent. In many cases, the elution process is used to separate, or resolve, unwanted species from the desired protein. The unwanted species may bind less tightly to the adsorbent and will be eluted prior to the product. In other cases, undesirable species bind more tightly to the adsorbent and will remain bound after the product is eluted. This is usually the case when HIC is used to separate protein aggregate species, which bind more tightly to the adsorbent than the desired protein monomer species. During the elution step, a portion (or fraction) of the eluate may contain highly purified product, while fractions before and after contain higher levels of undesirable impurities. A schematic of an elution process (during a gradient elution) is shown in Fig. 5.3. Figure 5.3 illustrates that the column effluent collected during the elution step may need to be fractionated in order to achieve acceptable product purity when using HIC. The elution process can be done using either a stepwise (isocratic) or a gradient approach. The four most common methods (listed from most common to least common) used to elute the bound protein include the following: i. Decrease in the salt concentration (relative to the binding conditions). A decrease in the salt concentration will decrease the strength of hydrophobic interactions between the protein and the ligand, and the protein will be desorbed and eluted from the column. ii. Addition of organic solvents. Addition of an organic solvent (such as ethylene or propylene glycol) changes the solvent polarity, which disrupts the hydrophobic interaction.
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Figure 5.3 Schematic chromatogram showing a gradient elution of a protein mixture using hydrophobic interaction chromatography. In the diagram, the salt concentration is linearly decreased (from high salt to low salt), which results in elution of both impurities and the target protein.
iii. Increase in the salt concentration (using a chaotropic salt). Addition of a chaotropic salt will disrupt the hydrophobic interaction. iv. Detergent addition. Detergents are used as protein displacers, and have been used mainly for the purification of membrane proteins when using HIC ( Janson and Ryde´n, 1997, see also Explanatory Chapter: Choosing the right detergent). This most common approach used to elute proteins from HIC adsorbents is by lowering the salt concentration during the elution step. This should be the first method that is attempted when using HIC for purification of a new protein compound. The other approaches described above have the disadvantage that an additional component (such as a chaotropic salt or an organic solvent) needs to be added, which may impact protein stability. However, such agents may be required in order to effectively elute a strongly bound protein species from the adsorbent. Each protein must be evaluated case by case to determine which elution method is appropriate. The HIC adsorbent used in the purification may also influence which elution method is effective.
7.3. Gradient elution Gradient elutions are an extremely effective method useful for screening different HIC adsorbents in protein purification. During the gradient elution
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process, the salt concentration is decreased gradually (in a linear fashion) from a high salt concentration to a low salt concentration over a defined volume. During the initial screening of a bound compound on an adsorbent, the salt concentration may be decreased to as low as 0 mM to determine the salt concentration when the product elutes. As a starting point, a typical gradient elution process is performed over ten (10) column volumes, during which fractions are collected and evaluated for product purity. The gradient in salt concentration may be decreased (performed over a larger volume) in order to improve protein resolution (Yamamoto et al., 1988). In the event that the protein remains bound to the adsorbent following the gradient elution process, this may indicate that either a weaker lyotropic salt should be selected to bind the protein to the adsorbent, or that a stronger elution condition is required to elute the protein. Stronger elution solutions may include the use of an organic solvent, including propylene glycol, ethylene glycol, acetonitrile, or ethanol. Alternatively, an adsorbent with weaker hydrophobic binding strength may need to be selected to decrease the strength of hydrophobic interaction and achieve an acceptable product recovery.
7.4. Stepwise (isocratic) elution After identifying the appropriate adsorbent and salt concentration to effectively elute the protein of interest, an isocratic elution can be used if desired. An advantage of using isocratic elution is its simplicity – it requires a simple switch in the inlet buffer (from a high to a low salt concentration). Use of an isocratic elution is a preferable approach to simplify the equipment requirements, as gradient elution requires multiple pumps and additional process control to generate a linear change in the buffer salt concentration.
8. STEP 4 ADSORBENT REGENERATION AND SANITIZATION 8.1. Overview The HIC adsorbent will be washed, regenerated, and placed into storage buffer for reuse.
8.2. Duration 3–6 h
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4.1 Wash the column using 3–5 column volumes of regeneration buffer (e.g., 6-M guanidine hydrochloride, or 20–100% ethanol or methanol) over a period of 1–2 h. 4.2 Wash the column using 3–5 column volumes of sanitization buffer (e.g., 1-M NaOH) over a period of 1–2 h. 4.3 Wash the column using 3–5 column volumes of storage buffer (e.g., 20% ethanol or 0.1M NaOH) over a period of 1–2h. Comments: HIC adsorbents are reusable for multiple cycles and have a relatively long lifetime before having to be replaced. However, adsorbents must be cleaned and regenerated between uses in order to ensure reproducible performance over many cycles. The adsorbent manufacturers provide regeneration procedures for the adsorbents, which can be consulted prior to use. In general, the cleaning procedures depend upon the stability of the base matrix and the hydrophobic ligand. For strongly bound proteins, 6-M guanidine hydrochloride is often recommended. If detergents have been used during the process, ethanol or methanol can be used as part of the regeneration procedure (GE Healthcare, 2006). For sanitization, a caustic solution (1.0-M NaOH) can be used for most of the adsorbents (with the exception of silica). The adsorbent manufacturer should also provide information on the appropriate storage conditions. In a typical process, the HIC column is exposed to subsequent regeneration and sanitization solutions for 1–2 h for each step, using 3–5 column volumes. Following the regeneration and sanitization steps, a storage solution should be selected that prevents microbial growth, but does not impact ligand or base matrix stability. For the storage step, 3–5 column volumes of storage solution should be passed through the column to ensure that the column is properly equilibrated prior to storage.
REFERENCES Referenced Literature Ben-Naim, A. (1980). Hydrophobic Interactions. New York: Plenum Press. Chotia, C. (1976). Surface of monomelic proteins. Journal of Molecular Biology, 105, 1–12. Fausnaugh, J. L., & Regnier, F. E. (1986). Solute and mobil phase contributions to retention in hydrophobic interaction chromatography of proteins. Journal of Chromatography, 359, 131–146. GE Healthcare (2006). Data File No. 18-1127-63 AC. Hjerte´n, S., Rosengren, J., & Pa˚hlman, S. (1974). Hydrophobic interaction chromatography. Journal of Chromatography, 101, 281–288. Hofmeister, F. (1988). On regularities in the albumin precipitation reactions with salts and their relationship to physiological behavior. Archiv for Experimentelle Pathologie und Pharmakologie, 24, 247–260.
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Janson, J.-C., & Ryde´n, L. (Eds.), (1997). Protein Purification: Principles, High-Resolution Methods, and Applications (2nd edn., p. 284). Weinheim: Wiley-VCH. Jones, D. D. (1975). Amino acid properties and side-chain orientation in proteins. Journal of Theoretical Biology, 50, 167–183. Krigbaum, W. R., & Komoriya, A. (1979). Local interactions as a structure determinant for protein molecules. Biochimica et Biophysica Acta, 576, 204–248. Manavalan, P., & Ponnuswamy, P. K. (1978). Hydrophobic character of amino acid residues in globular proteins. Nature, 275, 673–674. Melander, W., & Horvath, C. (1977). Salt effects on hydrophobic interactions in precipitation and chromatography of proteins: An interpretation of the lyotropic series. Archives of Biochemistry and Biophysics, 183, 200–215. Nozaki, Y., & Tanford, C. (1971). The solubility of amino acids and two glycine peptides in aqueous ethanol and dioxane solutions. Establishment of a hydrophobicity scale. Journal of Biological Chemistry, 246, 2211–2217. Pa˚hlman, S., Rosengren, J., & Hjerten, S. (1977). Hydrophobic interaction chromatography on uncharged sepharose derivatives. Journal of Chromatography, 131, 99–108. Porath, J., & Larsson, B. (1978). Charge-transfer and water-mediated chromatography. I: Electron-acceptor ligands on cross-linked dextran Journal of Chromatography, 155, 47–68. Rose, G. D., Geselowitz, A. R., Lesser, G. J., Lee, R. H., & Zehfus, M. H. (1985). Hydrophobicity of amino acid residues in globular proteins. Science, 229, 834–838. Tanford, C. (1962). Contribution of hydrophobic interactions to the stability of the globular conformation of proteins. Journal of American Chemical Society, 84, 4240–4247. Tanford, C. (1980). The Hydrophobic Effect (2nd edn.). New York: Wiley. Ulbrich, V., Makes, J., & Jurecek, M. (1964). Identification of giycidyl ethers. Bis(phenyl-) and bis (a-naphthylurethans) of glycerol a-alkyl (aryl)ethers. Collection of Czechoslovak Chemical Communications, 29, 1466–1475. Wertz, D. H., & Scheraga, H. A. (1978). Influence of water on protein structure. Macromolecules, 11, 9–15. Yamamoto, S., Nakanishi, K., & Matsuno, R. (1988). Ion-Exchange Chromatography of Proteins. New York: Mercel Dekkar. Zimmerman, J. M., Eliezer, N., & Simha, R. (1968). The characterization of amino acid sequences in proteins by statistical methods. Journal of Theoretical Biology, 21(2), 170–201.
SOURCE REFERENCES Burgess, R., & Deutshcer, M. (Eds.), (2009). Methods in Enzymology, Guide to Protein Purification (2nd edn, pp. 405–413). New York: Elsevier, vol. 463. Hjerte´n, S., Yao, K., Eriksson, K.-O., & Johansson, B. (1986). Gradient and isocratic high performance hydrophobic interaction chromatography of proteins on agarose columns. Journal of Chromatography, 359, 99–109.
Referenced Protocols in Methods Navigator Using ion exchange chromatography to purify a recombinantly expressed protein. Purification of His-tagged proteins. Affinity purification of a recombinant protein expressed as a fusion with the maltose-binding protein (MBP) tag. Purification of GST-tagged proteins. Protein Affinity Purification using Intein/Chitin Binding Protein Tags. Immunoaffinity purification of proteins. Strep-tagged protein purification. Explanatory Chapter: Choosing the right detergent.
