Methods and Experiments in Biochemistry

1. Methods and Experiments in Biochemistry Protein Purification Geen-Dong Chang gdchang@ccms.ntu.edu.tw. N402, Institute of Biochemical Sciences, N.T.U. Edited by Janway Chen

2. Protein Purification ● Choice of raw materials: concentration, availability, cost and stability ● Extraction methods: solution, tissues, or organisms ● Extraction medium ● Purification methods ● Sequence of chromatography Edited by Janway Chen

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4. Resolution is achieved by the selectivity of the technique and the efficiency of the chromatographic matrix in producing narrow peaks. In general, resolution is most difficult to achieve in the final stages of purification when impurities and target protein are likely to have very similar properties. The high selectivity of affinity chromatography typically gives a high resolution result. Capacity, in the simple model shown, refers to the amount of target protein that can be loaded during purification. In some cases the amount of sample that can be loaded will be limited by volume (as in gel filtration) or by large amounts of contaminants, rather than by the amount of the target protein. Since affinity chromatography is a binding technique the separation is unaffected by sample volume as long as the correct binding conditions are maintained during sample application and the total amount of target protein loaded onto the column does not exceed the binding capacity of the affinity medium. Speed is most important at the beginning of purification where contaminants such as proteases must be removed as quickly as possible. Modern affinity matrices enable high flow rates to be used for sample application as well as washing and reequilibration steps. For each application a flow rate can be selected to achieve an optimal balance between efficient binding and elution of the target protein and a fast separation. Recovery becomes increasingly important as the purification proceeds because of the increased value of the purified product. Recovery is influenced by destructive processes in the sample and by unfavourable conditions on the column. Affinity media provided with optimized separation protocols can give extremely high recoveries of target protein. Edited by Janway Chen

5. Mechanical Liquidd shear Solid shear Non-mechanical Desiccation Lysis

6. Extraction Medium • pH • Buffers and salts • Detergents and chaotropic agents (membrane proteins); guanidine hydrochloride, sodium thiocyanate, sodium iodide • Reducing agents: 2-mercaptoethanol, 1,4-dithiothreitol and 1,4-dithioerythritol • Chelators (EDTA, EGTA) or metal ions (calcium, magnesium) • Proteolytic inhibitors: serine proteases, metallorpoteases, cysteine proteases, and aspartic proteases • Bacteriostatics: 1 mM sodium azide, 0.005% merthiolate Edited by Janway Chen

7. Proteolytic inhibitors Edited by Janway Chen

8. Precipitation ● Salting out:  hydrophobic interaction (antichaotropic); ammonium sulfate, sodium sulfate ● Organic solvents:  water activity; ethanol, acetone ● Organic polymers:  water activity; polyethylene glycol ● pH adjustment: lowest solubility at pI points, extreme pH ● Temperature: heat-stable proteins Precipitation agent

9. The Hofmeister series of ions Increasing precipitation (salting-out) effect ● Anions: PO43-, SO42-, CH3COO-, Cl-, Br-, NO3-, ClO4-, I-, SCN- ● Cations: NH4+, Rb+, K+, Na+, Cs+, Li+, Mg2+, Ca2+, Ba2+ Increasing chaotropic (salting-in) effect Edited by Janway Chen

10. Order of chromatographic steps ● Sample volume ● Protein concentration and viscosity of the sample ● Degree of purity of the protein product needed ● Presence of nucleic acids, pyrogens and proteolytic enzymes in the samples ● To minimize buffer changes and concentration steps ● Consideration of cost, capacity, resolution, speed and recovery Edited by Janway Chen

11. Ligand-protein interactions ● Ion-ion or ion-dipole bonds ● Hydrogen bonds ● van der Waals forces ● Aromatic or - interactions ● Hydrophobic interaction ● Covalent bonds Edited by Janway Chen

12. ● Biomolecules are purified using purification techniques that separate according to differences in specific properties, as shown below. Edited by Janway Chen

13. Summary of chromatographic techniques commonly used in protein purification.

15. Stationary phasePorous matrix + imbibed immobile solvent + functional groups ● Cross-linked dextran ● Cross-linked polyacrylamide ● Porous glass ● Silica ● Polystyrene ● Polymethacrylate ● Ceramics Edited by Janway Chen

16. Chromatographic development ● Isocratic elution: same buffer used in equilibration and elution; gel filtration ● Gradient elution: stepwise, linear gradient, most column chromatography ● Displacement elution; affinity column chromatography Edited by Janway Chen

17. Column Capacity (Q) ● Nominal binding capacity: amounts of functional group determined by acid-base titration ● Dynamic (functional) capacity: flow rate dependent

18. Gel filtration or size exclusion chromatography (SEC) separates molecules based on their size. The gel media consists of spherical beads containing pores of a specific size distribution. Separation occurs when molecules of different sizes are included or excluded from the pores within the matrix. Small molecules diffuse into the pores and their flow through the column is retarded, while large molecules do not enter the pores and are eluted in the column's void volume. Consequently, molecules separate based on their size as they pass through the column and are eluted in order of decreasing molecular weight. Operating conditions and gel selection depend on the application and the desired resolution. Two methods used in size exclusion chromatography are group separation, including desalting and buffer exchange, and fractionation. In desalting, the molecule of interest is eluted in the void volume, while smaller molecules are retained in the gel pores. To obtain the desired separation, the gel should have an exclusion limit smaller than the molecule of interest. In fractionation, molecules of varying molecular weights are separated within the gel matrix. With this method, the molecules of interest should fall within the fractionation range of the gel. Common applications include the fractionation and molecular weight determination of proteins, nucleic acid separations, plasmid purification, and polysaccharide fractionation. Resolution depends on the particle size, pore size, flow rate, column length and diameter, and sample volume. Generally, the highest resolution is obtained with low flow rates (2–10 cm/hr), long narrow columns, small particle size gels, small sample volumes (1–5% of the total bed volume), a 2-fold difference in molecular weight and a sample viscosity that is the same as the eluant. For desalting, the sample volume can be as much as 30–40% of the total bed volume, and shorter wider columns maybe used. Edited by Janway Chen

19. ● Gel filtration is largely independent of sample concentration. The volume of the sample relative to the bed volume is far more important. For analytical purposes the sample should not be larger than 1-5% of the bed volume, whereas for desalting the sample can be as large as 30-35% of the bed volume. The viscosity of the sample may limit the concentration of sample which can be used. Viscous samples may be diluted to decrease the viscosity. It may be possible to achieve better results by applying viscous samples at a lower flow rate. The sample should be clear, and completely dissolved in running buffer, without particles or solid contaminants. Filtration of samples will increase column life. If, due to the nature of the sample, it is not possible to filter it, the sample should be centrifuged until it is clear. Figure 7 shows the hypothetical effects of various chromatographic conditions.

20. cellulose Acrylamide-bis-acrylamide dextran agarose

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24. Selectivity curves for Sephacryl HR in phosphate buffer (0.05M, pH7.0) containing NaCl (0.15M). Globular protein Dextran standards M.W. M.W.

25. + + + + + + + + + + + + + + + + + + + + + + + + + Starting buffer counter-ions Substances to be separated Gradient ions The principles of ion exchange chromatography (salt gradient elution). Start of desorption End of desorption Starting conditions Adsorption of sample substances Regeneration Edited by Janway Chen

26. Ion exchange chromatography is based on the binding of charged sample molecules to oppositely charged groups attached to an insoluble matrix. Substances are bound to ion exchangers when they carry a net charge opposite to that of the ion exchanger. This binding is electrostatic and reversible. The pH value at which a biomolecule carries no net charge is called the isoelectric point (pI). When exposed to a pH below its pI, the biomolecule will carry a positive net charge and will bind to a cation exchanger (SP and CM). At pH’s above its pI the biomolecule will carry a negative net charge and will bind to an anion exchanger (Q, DEAE and ANX). If the sample components are most stable below their pI’s, a cation exchanger should be used. If they are most stable above their pI’s, an anion exchanger is used. If stability is high over a wide pH range on both side of the pI, either type of ion exchanger can be used. Weak ion exchangers have a limited pH working range. Information on the pI and how the net charge on the molecule varies with pH gives valuable information regarding the choice of starting conditions. Electrophoretic titration curves enable the determination of the charge/pH relationship for the molecules present across the pH range of interest. Edited by Janway Chen

27. ● Charged amino acid side chains and some other groups in proteins.

29. Functional group used in ion exchangers.

30. DEAE-sepharose CL-4B CM-sepharose CL-4B Edited by Janway Chen

31. Q-sepharose fast flow S-sepharose fast flow Edited by Janway Chen

32. Test-tube methods for selecting ion exchange conditions. Determine optimum pH Determine salt conc. For binding + elution Determine available capacity Edited by Janway Chen

33. 1. Set up a series of buffers with different pH’s, in the range 4 – 8 (SP, CM) or 5 – 9 (Q, DEAE, ANX), with 0.5 – 1 pH unit intervals between each buffer. Make one series with 1 M NaCl included in the buffers (elution buffer) and the other without NaCl (start buffer). 2. Equilibrate the column with start buffer. 3. Adjust the sample to the chosen start buffer. 4. Apply a constant known amount of the sample at 1 ml/min. Collect the eluate. 5. Wash with at least 5x volume of start buffer or until no material appears in effluent. Collect the eluate. 6. Elute bound material with elution buffer. 3 – 5 x volume is usually sufficient but other volumes may be required dependent on the exact experimental conditions. Collect the eluate. 7. Analyse all eluates (by activity assay for example) and determine the purity and the amount bound to the column. 8. Perform steps 2 – 7 for the next buffer pH. 9. Decide which pH should be used for the selected purification strategy. 10. To decide on starting ionic strength conditions, a similar screening is done, but the buffer pH is held constant and the salt concentration is varied in the interval 0 – 0.5 M, with intervals of 0.05 – 0.1 M salt between each buffer.

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36. Choice of gradient type 1. Stepwise gradients are easy to produce and require minimal equipment. Eluted peaks are very sharp and elution volumes minimal. However, care must be exercised in the design of the steps and the interpretation of results for substances eluted by a sharp change in pH or small differences in ionic strength. Peaks tend to have sharp fronts and pronounced tailing since they frequently contain more than one component. 2. Continuous salt gradients are the most frequently used type of elution. Many types of gradient forming systems are available. Two buffers of differing ionic strength, the start and elution buffer (start buffer + 1 M NaCl or higher buffer salt concentration), are mixed together and if the volume ratio is changed linearly, the ionic strength changes linearly. 3. Another, but less common, method to desorb bound material is to increase (SP and CM) or decrease (Q, DEAE and ANX) the pH of the eluent. Continuous pH gradients are difficult to produce at constant ionic strength, since simultaneous changes in ionic strength, although small, also occur (buffering capacities are pH dependent). In the case of pH gradients using weak ion exchangers (CM, DEAE and ANX) the buffer may have to titrate the ion exchanger and there will be a short period of re-equilibration before the new pH is reached. Edited by Janway Chen

38. Hydrophobic Interaction Chromatography Substances are separated on the basis of their varying strengths of hydrophobic interactions with hydrophobic ligands immobilized to an uncharged matrix. This technique is usually performed with moderately high concentrations of salts in the start buffer (salt promoted adsorption). Elution is achieved by a linear or stepwise decrease in salt concentration. Factor affecting HIC The type of ligand, the degree of substitution, the pH and the type and concentration of salt used during the adsorption stage have a profound effect on the overall performance (e.g. selectivity and capacity) of a HIC matrix. Other factors that affect HIC are temperature, detergents, polarity of solvents, type of matrix and ligand coupling chemistry. The type of immobilised ligand determines primarily the selectivity of the HIC adsorbent. In general, HIC media fall into two groups, depending on their interactions with sample components. Straight alkyl chains (butyl, octyl) show a ”pure” hydrophobic character, while aryl ligands (phenyl) show a mixed mode behaviour, where both aromatic and hydrophobic interactions as well as lack of charge play simultaneous roles. The choice of ligand must be determined empirically through screening experiments for each individual separation problem. The protein binding capacity of HIC adsorbents increases with increased degree of substitution up to a certain level and then levels off. Simultaneously, the strength of the interaction increases, which may lead to difficulties in the elution of bound compounds. This potential problem has been addressed by the development of matrices with different levels of ligand density. The solvent is one of the most important parameters which influences capacity and selectivity in HIC. In general, the adsorption process is more selective than the desorption process. It is therefore important to optimize the starting buffer with respect to pH, type of solvent, type of salt and concentration of salt. The addition of various ”salting-out” salts to the sample promotes ligand-protein interactions in HIC. As the concentration of salt is increased, the amount of bound protein increases up to the precipitation point for the protein. Each type of salt differs in its ability to promote hydrophobic interactions and it may be worthwhile testing several salts. The most commonly used salts are (NH 4 ) 2 SO 4 , Na 2 SO 4 , NaCl, KCl and CH 3 COONH 4 .

39. ● Due to instability, ammonium sulphate is not suitable when working at pH values above 8.0. Sodium sulphate is also a very good salting-out agent but protein solubility problems may exclude its use at high concentrations. The effect of pH in HIC is not straightforward. In general, an increase in pH weakens hydrophobic interactions. Retention of proteins changes more drastically at pH values above 8.5 or below 5.0 than in the range 5.0-8.5. These findings suggest that pH is an important separation parameter and it is advisable to check the applicability to the particular problem at hand. Increasing the temperature enhances hydrophobic interactions in most cases. One should thus be aware that a process developed at room temperature might not be reproducible in the cold room and vice versa. Sometimes it is necessary to weaken the protein-ligand interactions by including different additives. Commonly used are water-miscible alcohols (propanol, ethylene glycol), detergents (SDS) and solutions of chaotrophic salts (lithium perchlorate, urea, guanidine hydrochloride). Elution with linear descending gradients A linear decrease of the salt concentration is the most frequently used type of elution in hydrophobic interaction chromatography. Recommended buffers are 50 mM sodium phosphate, 1.0 M ammonium sulphate, pH 7.0 as start buffer and 50 mM sodium phosphate, pH 7.0 as elution buffer. Continuous gradients can be prepared in different ways depending on available equipment. Elution with stepwise descending gradients Stepwise elution is the sequential use of the same buffer at different ionic strengths. It is technically simple and fast and suitable for syringe operation. It is often used for sample concentration and sample clean up. Stepwise elution gives small peak volumes and the resolution depends on the difference in elution power between each step. When stepwise elution is applied, one has to keep in mind the danger of artifactual peaks when a subsequent step is executed too early after a tailing peak. For this reason it is recommended to start with a continuous gradient to characterise the sample and its chromatographic behaviour.

40. Affinity chromatography separates proteins on the basis of a reversible interaction between a protein (or group of proteins) and a specific ligand coupled to a chromatographic matrix. The technique offers high selectivity, hence high resolution, and usually high capacity for the protein(s) of interest. Purification can be in the order of several thousand-fold and recoveries of active material are generally very high. Affinity chromatography is unique in purification technology since it is the only technique that enables the purification of a biomolecule on the basis of its biological function or individual chemical structure. Purification that would otherwise be time-consuming, difficult or even impossible using other techniques can often be easily achieved with affinity chromatography. The technique can be used to separate active biomolecules from denatured or functionally different forms, to isolate pure substances present at low concentration in large volumes of crude sample and also to remove specific contaminants. Biological interactions between ligand and target molecule can be a result of electrostatic or hydrophobic interactions, van der Waals' forces and/or hydrogen bonding. To elute the target molecule from the affinity medium the interaction can be reversed, either specifically using a competitive ligand, or non-specifically, by changing the pH, ionic strength or polarity. Edited by Janway Chen

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43. Common terms in affinity chromatography Matrix: for ligand attachment. Matrix should be chemically and physically inert. Spacer arm: used to improve binding between ligand and target molecule by overcoming any effects of steric hindrance. Ligand: molecule that binds reversibly to a specific target molecule or group of target molecules. Binding: buffer conditions are optimized to ensure that the target molecules interact effectively with the ligand and are retained by the affinity medium as all other molecules wash through the column. Elution: buffer conditions are changed to reverse (weaken) the interaction between the target molecules and the ligand so that the target molecules can be eluted from the column. Wash: buffer conditions that wash unbound substances from the column without eluting the target molecules or that re-equilibrate the column back to the starting conditions (in most cases the binding buffer is used as a wash buffer). Ligand coupling: covalent attachment of a ligand to a suitable pre-activated matrix to create an affinity medium. Pre-activated matrices: matrices which have been chemically modified to facilitate the coupling of specific types of ligand. Edited by Janway Chen

45. pH elution A change in pH alters the degree of ionization of charged groups on the ligand and/or the bound protein. This change may affect the binding sites directly, reducing their affinity, or cause indirect changes in affinity by alterations in conformation. A step decrease in pH is the most common way to elute bound substances. The chemical stability of the matrix, ligand and target protein determines the limit of pH that may be used. If low pH must be used, collect fractions into neutralization buffer such as 1 M Tris-HCl,pH 9 (60–200 µl per ml eluted fraction) to return the fraction to a neutral pH. The column should also be re-equilibrated to neutral pH immediately. Ionic strength elution The exact mechanism for elution by changes in ionic strength will depend upon the specific interaction between the ligand and target protein. This is a mild elution using a buffer with increased ionic strength (usually NaCl), applied as a linear gradient or in steps. Enzymes usually elute at a concentration of 1 M NaCl or less. Edited by Janway Chen

46. Competitive elution Selective eluents are often used to separate substances on a group specific medium or when the binding affinity of the ligand/target protein interaction is relatively high. The eluting agent competes either for binding to the target protein or for binding to the ligand. Substances may be eluted either by a concentration gradient of a single eluent or by pulse elution. When working with competitive elution the concentration of competing compound should be similar to the concentration of the coupled ligand. However, if the free competing compound binds more weakly than the ligand to the target molecule, use a concentration ten-fold higher than that of the ligand. Reduced polarity of eluent Conditions are used to lower the polarity of the eluent promote elution without inactivating the eluted substances. Dioxane (up to 10%) or ethylene glycol (up to 50%) are typical of this type of eluent. Chaotropic eluents If other elution methods fail, deforming buffers, which alter the structure of proteins, can be used, e.g. chaotropic agents such as guanidine hydrochloride or urea. Chaotropes should be avoided whenever possible since they are likely to denature the eluted protein. Edited by Janway Chen

47. ● Step elution ● Gradient elution Edited by Janway Chen

48. ● Gradient elution of a (His)6 fusion protein. Edited by Janway Chen

49. ● Scouting for optimal elution pH of a monoclonal IgG3 from HiTrap rProtein A FE, using a pH gradient. Edited by Janway Chen