Experiments on the theory and application of hydrophobic interaction chromatography in protein purification
Experiments on the theory and application of hydrophobic interaction chromatography in protein purification
Hydrophobic interaction chromatography (HIC) is a valuable tool in protein purification technology. HIC is used for protein purification on a wide range of scales - both analytical and preparative - and can be used to remove a wide range of impurities present in solution, including unwanted product-related impurities. In particular, HIC can be used to remove polymeric sarcophosphates from product towels, which are generally not as effective as the property of transitions. In this chapter, we will introduce the HIC consultant's theory and how to purify protein from hydronic towel. With the theoretical background, the latest technology in the field of HIC adsorbents is described, including commonly used and commercially available adsorbents. Basic procedures for the application of HIC adsorbents are then described to provide the reader with a starting point for protein purification with HIC.
Authors: Burgess et al., Translator: Chen Wei, This experiment is from the "Guide to Protein Purification".
Operation method
Experiments on the theory and application of hydrophobic interaction chromatography in protein purification Move H I C Adsorbent Usage 1. Introduction H IC adsorbents are used directly in the purification of protein complexes. The purification process consists of a series of steps as follows, which are described in some detail in this section. The hydrophobicity of a given protein is usually unknown, so screening of several adsorbents is required before a final adsorbent can be selected. The screening process is to determine the strength of binding of the adsorbent to protein and to decide which candidate adsorbents can be used for protein purification. Preliminary screening for adsorbent hydrophobicity is used to determine the hydrophobicity of the compound and which adsorbents will be effective in purifying the protein, and should include a broad range of adsorbents with a wide range of hydrophobic binding properties. Before the sample is loaded onto the column, the salt concentration of the protein mixture (the sample to be purified with the HIC absorbent) must be increased by using a high salt concentration buffer in order to bind the target protein to the absorbent. Since protein may precipitate at high salt concentrations, the solubility of the protein complex in a given salt solution should be evaluated prior to selecting the salt solution. The pH of the buffer does not show a general trend in the effect of the protein binding to the HIC absorber, but can influence the strength of the interaction (Hjertte etal., 1974). The pH of the buffer should be chosen to ensure that the protein and the adsorbent are stabilized in the environment (e.g., avoid extreme pH solutions). During the steps for adjusting the protein sample, the salt concentration can be 0.5 to 2.0 m o l l . The salt concentration can be in the range of 0.5 to 2.0 mOl/L and can be increased to ensure that the protein is effectively bound to the absorbent. The salt concentration required for binding of protein to an absorbent depends to a large extent on the choice of salt type, as described in section 3.2. In most cases, the selection of the appropriate salt concentration for protein binding to an absorbent requires relevant pre-testing. Before the protein feedstock is sampled, the chromatography column should first be equilibrated with a high salt concentration buffer of similar composition (salt concentration) and P h to the feedstock to ensure that the proteins can bind tightly to the adsorbent. This step is called equilibration. After equilibration, the adjusted material (containing the target protein) is upsampled onto the HIC column at a suitable flow rate. After sampling, the column is usually washed with an equilibration buffer and the target protein is eluted. Prior to elution, an additional wash step is performed to remove unwanted protein impurities. The salt concentration used in this wash step is intermediate - less than that used in the sample step, but more than that used in the elution step. After the protein is bound to the adsorbent, the target protein must be eluted and the post-column effluent collected. In most cases, the elution process is used to separate or remove unwanted proteins from the target protein. The unwanted proteins may not be as tightly bound to the adsorbent and may be eluted before the target protein. In other cases, the unwanted protein will bind more tightly to the adsorbent and will remain bound to the adsorbent after the target protein has been eluted. This usually occurs when H I C separates protein multimers, where the target protein multimer is more tightly bound to the adsorbent than the monomer is bound to the adsorbent. During the elution step, the effluent fraction may contain high purity The elution process can be performed in either a stepwise (isocratic) or gradient fashion. The four most common methods of eluting bound proteins (in order from most to least common) are described below. (1) Reducing the salt concentration (relative to the binding conditions). A decrease in salt concentration correspondingly decreases the hydrophobic interaction between the protein and the ligand, and the protein is adsorbed and eluted from the column. (2) Addition of organic solvents. The addition of organic solvents (e.g., ethylene or propylene glycol) can change the polarity of the solution, thus interfering with the hydrophobic interaction. (3) Increase the salt concentration (using free-liquid salt). The addition of free-liquid salt can interfere with the hydrophobic effect. (4) Addition of detergent. Decontaminants can be used as protein displacing agents, mainly for H IC purification of membrane proteins (Jansonand R y d e n , 1997). The most common method of eluting proteins from the H I C adsorbent in the elution step is to reduce the salt concentration. This method is likewise used first when purifying new protein fractions with H I C . The disadvantage of the other methods described above is that additional substances (e.g., leaving salts and organic solvents) are required, which may affect the stability of the protein. However, these reagents are required for the elution of proteins that are more tightly bound to the adsorbent. Each protein must be evaluated individually to determine the appropriate elution method to use. The H I C adsorbent used in the purification process may also affect the effectiveness of the elution method. Gradient elution is a very effective method for screening different HIC adsorbents in protein purification. In gradient elution, the salt concentration decreases from high to low (linearly) in a set volume. For the initial screening of protein-bound adsorbents, the salt concentration may even be reduced to 0 mmol/L in order to determine the salt concentration at which the product will elute. A typical gradient elution typically starts with 10 column volumes of elution buffer, and the effluent fraction is collected and evaluated for product purity. Reducing the salt concentration of the gradient (when performing larger volumes of elution) is intended to improve the resolution of the protein separation (Y a m a m 0-to et al., 1988). After gradient elution, proteins are still in the adsorbent-bound state, indicating that weaker labile salts should be used to bind proteins to the adsorbent or stronger elution conditions should be selected for protein elution. Stronger elution conditions include the use of organic solvents. Organic solvents that are not easily combustible (e.g., propylene or ethylene glycol) are typically used in the preparative scale. Organic solvents used in analytical scale may be acetonitrile and ethanol. Similarly, for adsorbents with weak hydrophobic binding capacity, it may be necessary to select accordingly to attenuate the strength of the hydrophobic interaction. After determining the appropriate adsorbent and the salt concentration that will effectively elute the target protein, isocratic elution can also be used if desired. The advantage of using isocratic elution is its simplicity - only a simple change of delivery buffer (from high to low salt concentration) is required. Isocratic elution is the preferred method if the equipment requirements are to be simplified, whereas gradient elution requires equipment with multiple working pumps and control of the linear change in buffer salt concentration. H I C adsorbents are reusable after multiple cycles of chromatographic separation and have a relatively long service life. However, the adsorbent must also be cleaned and regenerated to ensure that its performance is reproducible after multiple cycles. Regeneration protocols are generally available from the supplier of the adsorbent and should be read carefully before use. In general, the cleaning protocol depends on the stability of the base medium and hydrophobic ligands. For proteins with high binding capacity, 6 mol/L guanidine hydrochloride is usually recommended. If a detergent has been used in a previous step, regeneration is done with ethanol or methanol (GE). For more product details, please visit Aladdin Scientific website.
Adsorbents may be purchased as pre-packed columns from an adsorbent supplier or may need to be filled by the user. Where a column is to be packed, the supplier's packing instructions should be consulted. The correct filling of the column can be verified by using a dimension that corresponds to the theoretical plate height of the column. Once packed, the sorbent supplier will usually provide recommendations for operating ranges, including permissible flow rate ranges, conditions for chromatographic operation, and methods for cleaning the column. Different adsorbents have different acceptable operating conditions, and the sales literature of the product should be carefully read before use.
product, but can also contain large amounts of unwanted protein impurities before or after it. A sketch of the elution process (in gradient elution) is shown in Figure 25.2. Figure 25.2 illustrates that when purifying by H IC, an aliquot of the post-column effluent should be collected during the elution step in order to obtain a product of suitable purity. The gradient elution process is described in further detail in Section 3.6 of this chapter.
Healthcare, 2006). Aggressive solutions (l.o mol/L NaOH) can be used for most adsorbents (except SiO2) during the cleaning process. Suppliers will also provide information on appropriate storage conditions. Adsorbent storage solutions should be selected on the basis of preventing the growth of microorganisms without affecting the stability of the matrix or base media.