Purification of enzymes.pptx

Purification of Enzymes/Proteins PURIFICATION OF ENZYMES Cell fractionation Nuclease enzymes Precipitation Basic substances Divalent metal ions Is...

Purification of Enzymes/Proteins PURIFICATION OF ENZYMES Cell fractionation Nuclease enzymes Precipitation Basic substances Divalent metal ions Isoelectric precipitation Sonication Preliminary purification Increasing the salt concentration Ammonium sulphate Ultrafiltration Polymer precipitation Chromatographic resin Laboratory-scale Purification Raise the temperature of the medium Preliminary purification procedures:  A preliminary purification step is usually included in the procedure used to extract an enzyme.  E.g.: Some degree of cell fractionation is often carried out before extraction, so that the relevant enzyme are extracted.  Also, the composition of the extraction medium is chosen so that the enzyme of interest is soluble but many others are not.  Extracted nucleic acids may cause problems by increasing the viscosity but they can be digested by the addition of nuclease enzymes or precipitated by the addition of basic substances (e.g. streptomycin, protamine, polyethyleneimine ), or divalent metal ions (e.g. MnCl2 or MgCl2).  Long fragments of nucleic acids can also be sheared by sonication.  All precipitates and cell debris are then removed by centrifugation and discarded.  The next stage of purification is usually to precipitate the enzyme of interest from solution, thus concentrating the protein and separating it from mono- and oligosaccharides, nucleotides, free amino acids etc. and from many other proteins which remain in solution.  Proteins show minimal solubility at their isoelectric point (pI) and can be precipitated by adjusting the pH to the pI of the enzyme.  However, the biological activity can be difficult to recover after isoelectric precipitation. Disadvantage of Isoelectric precipitation  The most enzyme-compatible method of precipitating proteins is by increasing the salt concentration: ammonium sulphate is often used for this purpose, because it is extremely soluble in water.  The salt concentration is usually increased in stages, the aim being to precipitate other proteins (which are then discarded) before precipitating the fraction containing the relevant enzyme. This is then redissolved in buffer, and residual ammonium sulphate removed by dialysis or size-exclusion chromatography.  Other methods suitable in laboratory-scale purification include ultrafiltration, polymer precipitation (e.g. polyethylene glycol, PEG) or the use of a chromatographic resin (e.g. ion exchange or hydrophobic interaction chromatography). In an industrial-scale purification, ultrafiltration, the use of stirred cells or expanded beds would be considered. Although a considerable degree of purification can be achieved by these procedures, many other proteins will still be present because of overlap of solubility ranges.  One possible way to remove some of these is to raise the temperature of the medium for a few minutes to a value where the enzymes being purified is known to be stable, but others might be denatured and precipitate from solution.  Enzymes are often particularly stable in the presence of their substrates, so the relevant substrate can be added to increase the effectiveness of this procedure. Further purification procedures: Partially purified crude extract can be treated in a variety of ways to increase the purification of the relevant enzyme. Chromatography based upon the partition (distribution) of material between two immiscible phases (a mobile phase and a stationary phase) represents the most popular collective technique for the purification of enzymes. There are a wide variety of protein-compatible resins available to which different functional groups can be attached to exploit the differences in protein structure.  There are a limited number of physical properties of proteins which can be utilized to enable the purification of an enzyme from a complex mixture.  Enzymes have different bio-specificity, surface charge, hydrophobicity, size, solubility and possibly post-translational modifications. Chromatographic purification techniques:  Ion exchange chromatography (IEX)  Hydrophobic interaction chromatography (HIC)  Reversed-phase HPLC  Hydrophobic charge induction chromatography (HCIC)  Mixed mode chromatography (MMC)  Affinity chromatography-Immobilized metal ion affinity chromatography (IMAC)  Immunoaffinity chromatography  Covalent chromatography  Size-exclusion chromatography (SEC) Purification techniques other than chromatography: Electrophoresis: zone electrophoresis, polyacrylamide gel electrophoresis (PAGE) Isoelectric focusing (IEF)  Ion exchange chromatography (IEX) Versatile technique with high binding capacity for the purification of enzymes, relying on the attraction of oppositely charged components.  At 1 pH unit above the pI of a protein, its surface will have an overall negative charge, so, at this pH, the protein will bind to a positively-charged resin, i.e. an anion exchange resin, such as one with a diethylaminoethyl (DEAE) functional group attached to a support such as cellulose. At 1 pH unit below the pI of a protein, its surface will have an overall positive charge so, at this pH, the protein will bind to a negatively-charged resin, such as one with a carboxymethyl (CM) functional group, attached to a suitable support. The binding can be reversed by lowering the pH in the case of anion exchange chromatography or by raising the pH in the case of anion exchange chromatography. Proteins will elute as the pH approaches their pI and the overall charge on the molecule approaches zero.  Many enzymes denature as they approach their isoelectric point, so bound proteins are usually eluted from IEX resins by raising the concentration of salt (0 - 2.0 M NaCl or KCl) in the eluting buffer.  The ions in the salt compete with the bound protein for the charge on the resin, and proteins elute according to their overall surface charge, with the lowest surface- charge proteins eluting first. The salt elution of enzymes bound to an IEX resin results in good recovery of activity, with usually a 10-fold increase in the purity.  Hydrophobic interaction chromatography (HIC) This technique depends on the fact that although amino acids with polar side chains predominate at the surface of enzymes, some non-polar ones will be present which can form hydrophobic interactions with column packings such as phenyl-Sepharose or octyl-Sepharose, particularly when the ionic strength is high (usually achieved with ammonium sulphate). Elution can be brought about by decreasing the ionic strength or introducing an organic solvent. Recovery of enzyme activity from HIC is usually good. In HIC, protein-binding to the ligand is determined by the concentration of lyotropic salts (e.g. ammonium sulphate).  Reversed-phase HPLC This technique also relies upon interaction between the hydrophobic areas of a protein and aliphatic ligands (e.g. C18, C12, C8 or C4) bonded to silica beads (3-10 mm diameter). The interaction between an enzyme and a reversed-phase resin is strong because there are more ligands bonded to the reversed-phase resin than to the HIC resin. However, the elution conditions of buffered aqueous and organic solvent mixtures rarely results in the recovery of biologically-active material.  Hydrophobic charge induction chromatography (HCIC) pH controls the binding of proteins to the hydrophobic ligand. The ligand is generally 4-mercapto-ethylpyridine (4-MEP) bonded to a modified cellulose resin. The resin was produced as an alternative to protein G for the purification of immunoglobulins and involves both 'pseudoaffinity' molecular recognition by interacting with the Fc region on the antibody and hydrophobic interactions at neutral pH. Elution is achieved by lowering the pH to around pH 5.0 and provides a means to enrich immunoglobulins essential free from contaminating albumin.  Mixed mode chromatography (MMC) Modified cellulose resin is one of a number (which include mercapto-benzimidazole sulphonic acid (MBI), hexylethylamino (HEA) and phenylpropylamino (PPA) resins produced by Pall; and Capto™ MMC produced by GE Healthcare) that operate in mixed mode chromatography. Protein interactions with MMC resins are not clear-cut and involve a mixture of different interactions. Potentially they provide the means to purify enzymes that are not easily resolved by other procedures.  Immobilized metal ion affinity chromatography (IMAC) Covalent bonding is characterized by an electron being shared with another atom, whereas, in coordinate (dative) bonding, both electrons are provided by one atom for another atom to share. The divalent metal Cu2+, Hg2+, Cd2+, Zn2+, Ni2+, Co2+ and Mn2+ can accept pairs of electron from electron-rich atoms to form coordinate bonds. At a physiological pH, some amino acids have functional groups with atoms that are rich in electrons including imidazole group of histidine, the thiol group of cysteine and the indole group of tryptophan, which can form dative bonds with metal ions. The amino acids with carboxylic acid residues (glutamic and aspartic acid) and the phosphate group on phosphorylated amino acids (e.g. phosphoserine, phosphothreonine and phosphotyrosine) can form coordinate bonds with trivalent metal ions at pH values around 3.0.  In IMAC, Resins typically have either iminodiacetate (IDA) or nitrilotriacetate (NTA) bound via a spacer-arm to Sepharose or agarose. These groups on the resin can bind di- or trivalent metal ions with coordinate bonds while leaving vacant orbitals which can then subsequently interact with the functional groups on amino acids (Histidine, cysteine and tryptophan). Elution of bound enzymes from the resin will depend upon the enzyme's stability and can be achieved by lowering the pH to below 5.0 or by displacement at pH 7.2 with increasing concentration of imidazole. The strength of the interaction between the bound protein and the bound metal ion will depend on the number and local environment of the histidine residues present.  The purification of recombinant proteins can be facilitated by the addition of a hexahistidine tag (6His) on either of the termini of the protein. The presence of the 6His tag results in strong binding of the recombinant protein to a metal charged IMAC resin, allowing the unwanted cellular proteins to be eluted prior to the elution of the recombinant protein. Hydroxyapatite (HA) is the crystalline form of calcium phosphate prepared by mixing calcium chloride and sodium phosphate and can be encapsulated in agarose (HA Ultrogel: Pall) or coated to a solid ceramic support (CHT: Bio-Rad). The surface of hydroxyapatite is comprised of pairs of positively-charged calcium ions, triplet clusters of negatively-charged phosphates and hydroxyl groups. Proteins added to HA show a mixed interaction with the resin which is difficult to predict but depends upon the protein's amino acid content and the pH and ionic strength of the buffer.  Affinity chromatography Enzymes are synthesized for a specific biological role and affinity chromatography exploits the bio- specificity of a protein. Affinity chromatography has the resolving power to isolate a pure enzyme from a complex mixture but the technique is rarely used in isolation, except in the purification of recombinant proteins. The bio-specific nature of these affinity techniques means that a high degree of purification can be achieved. On the other hand, it also means that a polymer-ligand complex prepared for the purification of one enzyme is limited to that particular application.  Immunoaffinity chromatography A closely related technique, in which immobilized ligand is an antibody specific for the enzyme which is being purified. The use of an immobilized monoclonal antibody as ligand gives even greater specificity than use of a more conventional antibody.  Another form of affinity chromatography involves the use of reactive triazine based dyes, e.g. Cibacron Blue F3G-A and dyes of the Procion series, as ligand.  Under alkaline conditions the chlorotriazine dye is linked, usually directly, to a matrix such as agarose by a triazine bond. The reaction involves a hydroxyl group on the matrix and a chloride on the dye.  Such immobilized dyes bind a wide range of NAD- and NADP-dependent dehydrogenases and other enzymes.  Covalent chromatography It is a separation technique developed to isolate thiol-containing proteins and peptides. A thiol residue attached to a resin can react with a protein's surface and/or active site sulphydryl groups to form a covalent mixed disulphide. The covalently-bound protein can then be eluted by a buffer containing reducing agents such as L-cysteine, 2-ME or dithiothreitol. THANK YOU