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Purification of Enzymes

Purification of Enzymes. Basic science: Its specificity for substrates Kinetic parameters Means of regulation Structure Mechanism of catalysis Understand the role of enzymes in more complex systems Use in medical and industrial applications. Initial Recovery of Protein.

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Purification of Enzymes

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  1. Purification of Enzymes Basic science: • Its specificity for substrates • Kinetic parameters • Means of regulation • Structure • Mechanism of catalysis • Understand the role of enzymes in more complex systems Use in medical and industrial applications

  2. Initial Recovery of Protein Intracellular or Extracellular? Cell Disruption Animal cells (no CW): • Potter homogenizer • Osmotic shock • Freeze-thaw cycles Plant cells (CW): • The Waring blender

  3. Microbial cells (CW):

  4. Removal of Whole Cells and Debris-1 • Centrifugation; batch vs. continuous-flow • 5000 g for 15 min for cells • 10 000g for 45 min for cell debris • High capital and running costs • Filtration; depth vs. membrane filters (0.1 -10 μm) • Separation of whole cells from fermantation media • Removal of whole cells and cell debris after cell disruption • Elimination of microbial species from product

  5. Removal of Whole Cells and Debris-2 • Aqueous two-phase partitioning • Gentle • Stabilization of proteins • Yield of protein activity high • Easy scale-up • Empirical

  6. Removal of Whole Cells and Debris-3 • Removal of nucleic acids • Liberation of large amounts of nucleic acids increases viscosity of cellular homogenate  difficult to process • Nucleic acid removal is especially important in the preparation of therapeutic proteins • Methods: precipitation (by polyethylenimine) or treatment with nucleases • Removal of lipids • It is a contaminant and can interfere with subsequent purification steps • Removal: Glass wool or a cloth of very fine mesh size

  7. Concentration and Primary Purification Large volumes of  Manageable dilute solution amount In laboratory scale: • Ultrafiltration • Precipitation • Ion-exchange • Dialysis • Freeze drying • Addition of dry Sephadex G-25

  8. Concentration by precipitation-1

  9. Concentration by precipitation-2 One of the oldest methods • Straight forward to perform • Uncomplicated equipment • High recovery of biological activity • Many precipitants are highly corrosive • Inefficient if initial protein concentration is low • Some precipitants are highy inflammable, some are expensive • Many precipitants must be disposed carefully • In many cases, precipitant must be removed totally

  10. Concentration by Ion-Exchange-1 • Isoelectronic point of proteins are different • (+)ly charged proteins  cation exchanger (CM) • (-)ly charged proteins  anion exchanger(DEAE) • Elution with a high ionic strength solution

  11. Concentration by Ion-Exchange-2 • Batchwise: • Extracellular proteins from fermentation broths or cell culture media • Cell debris from cell homogenates • Effective and relatively inexpensive • Easily regenerated • Considerable clarification of solution • Limited amount of protein purification

  12. Concentration by ultrafiltration-1 • Most widely applied method both in laboratory and industrial scale • Ultrafiltration membranes (pore diameters: 1 – 20 nm) • Molecular mass cut-off: 1 – 300 kDa (globular proteins) • Traditional materials: cellulose acetate and cellulose nitrate • Nowadays: PVC and polycarbonate • Concentration polarization can be a problem...

  13. Concentration by ultrafiltration-2 • Gentle • High recovery rates (even > 99 %) • Quick • Little ancillary equipment is needed • Some degree of protein purification • Susceptibility to rapid membrane clogging

  14. Column Chromatography Separation of different protein types from each other according to their differential partitioning between two phases: • A solid stationary phase • A liquid mobile phase Separation based on size and shape, overall charge, presence of surface hydrophobic groups, and ability to bind various ligands

  15. Different Chromatographic Techniques

  16. Gel Filtration Chromatography-1 • Also named as Size Exclusion Chromatography • Separation based on size and shape • Porous gel matrix in bead form is used: e.g. xlinked dextran, agarose, acrylamide • Large proteins come first....

  17. Gel Filtration Chromatography-2

  18. Gel Filtration Chromatography-3 EXAMPLES • Sephadex: dextran based, G-25 to G-200 • Sephacryl: allyl dextran based, more rigid and physically stable so suitable for large scale • Sepharose: agarose based, lack of physical stability • Bio-Gel P: acrylamide based A: agarose based • Fractogel: A copolymer, very high degree of mechanical stability

  19. Gel Filtration Chromatography-4 • Long chromatographic columns are needed (length/width = 25-40) • Rarely employed during the initial stages • Protein solution is significantly diluted • Column flowrates are often considerably lower

  20. Ion-Exchange Chromatography-1 FACTS • Proteins possess both (+) and (-) charges • At pH=7: • Aspartic and glutamic acid have negatively charged side groups • Lysine, arginine, histidine have positively charged side groups • pH of medium vs. pI of protein

  21. Ion-Exchange Chromatography-2 PRINCIPLE • Reversible electrostatic attraction of a charged molecule to a solid matrix possessing opposite charge • Elution is done by increasing salt concentration or changing pH

  22. Ion-Exchange Chromatography-3 • Single most popular chromatographic technique... • High level of resolution • Easy scale-up • Ease of usage • Easy column regeneration • One of the least expensive

  23. Ion-Exchange Chromatography-4 Inert, rigid and porous matrix materials are desirable EXAMPLES • Cellulose-based • Improved cellulose-based, e.g. diethylaminoethyl (DEAE) Sephacel • Sephadex; charged groups attached to Sephadex G-25 or G-50 • Based on polymers: Agarose and Sepharose • Alternative one: tentacle type

  24. Hydrophobic Interaction Chromatography-1 • 8 out 20 commonly found a.acids in proteins are classified as hydrophobic • In most proteins the majority of hydrophobic residues are buried inside the protein • Different proteins have different degree of hydrophobic surface

  25. Hydrophobic Interaction Chromatography-2 EXAMPLES • Most popular resins are hydrophobic group attached xlinked agarose gels • e.g. octyl- and phenyl-Sepharose gels

  26. Affinity Chromatography-1 • Described as the most powerful highly selective method • It relies on the ability of most proteins to bind specifically and reversibly to their ligands • Generally used in late purification steps

  27. Affinity Chromatography-2 • Biospecific affinity chromatography • General ligand approach: Cofactors (NAD+) or lectins • Specific ligand approach: enzyme substrates, substrate analogues or inhibitors, antibodies • Pseudoaffinity chromatography: e.g. Dye affinity chromatography

  28. Affinity Chromatography-3 Biospecific Affinity Chromatography • Choice of affinity ligand Specificity, reversible binding, stability • Choice of support matrix Stability, rigidity, inertness, porosity, derivatizable, inexpensive, reusable e.g. agarose, cellulose, silica and various organic polymers • Choice of chemical coupling technology nonhazardous, inexpensive, rapid. Spacer arm?

  29. Affinity Chromatography-4 • Increase in purity of over 1000-fold, with almost 100 % yields are reported (at least in lab scale) • Drastically reduce number of subsequent steps • Ligands are extremely expensive and often exhibit poor stability • Ligand coupling techniques are chemically complex, hazardous, time-consuming and costly • Leaching of ligand causes: • The reduction of system effectiveness • The presence of undesirable contaminant in product

  30. Affinity Chromatography-4 • Immunoaffinity purification • Polyclonal antibodies: low binding capacity, some other proteins can also bind • Monoclonal antibodies: monospecific • Relatively high cost technique • Antibody leakage may occur • Elution is difficult (e.g. glycine-HCl buffer with pH 2.2-2.8)

  31. Affinity Chromatography-5 • Lectin affinity chromatography • Lectins are a group of proteins synthesized by plants, vertebrates and some invertabrates (e.g. concanavalin A, soybean lectin) • In glycoprotein purification • Many lectins are expensive • Co-purification of glycoproteins • Little track record

  32. Affinity Chromatography-7 • Dye affinity chromatography Triazine dyes (e.g. cibacron blue F3G-A) are used • Dyes are available in bulk and relatively inexpensive • Chemical coupling is easy • Dye-matrix linkage is relatively resistant • The protein binding capacity is high • Elution is relatively easy • Textile dyes contain varying amount of impurities • Highly empirical

  33. Affinity Chromatography-8 • Metal chelate affinity chromatography • Iminodiacetic acid (IDA) • e.g. Ni, Cu, Zn, Fe • Basic groups, mostly side chain of His • Mostly used in recombinant protein purification

  34. Chromatofocussing • Separation based on isoelectic point of proteins • Pre-equilibrate a ion-exchange column at a pH • Pour slowly a buffer of different pH • Due to natural buffering capacity of exchanger, pH gradient will occur along the column • Usually a weak anion exchanger is used • Pre-equilibrated with high pH value • Pass a low pH value buffer

  35. High Pressure Liquid Chromatography-1 (HPLC) • Microparticulate stationary phase media of narrow diameter is used • Time for diffusion is reduced • Sample fractionation time is reduced • BUT pressure increases • Ideal support material • Mechanically & chemically stable • Low degree of non-specific adsorption • Reusable and inexpensive • Available in small size with narrow distribution • High degree of porosity • Silica gel, xlinked polystyrene are generally used

  36. High Pressure Liquid Chromatography-2 • Preparative HPLC Length: up to 80 cm, wider diameter • Analytical HPLC Length: 10-30 cm, diameter: 4-4.6 mm • Many small molecules can be purified by HPLC • In industrial scale, preparative HPLC is used in purification of insulin, interleukin-2

  37. High Pressure Liquid Chromatography-3 • Superiour resolution due to small particle size • Fast • High degree of automation • Cost • Capacity • Generally used for high value proteins intended for therapeutic use

  38. Fast Protein Liquid Chromatography(FPLC) • Operating pressure is significantly lower • Glass or inert plastic columns in stead of stainless steel • Economically more attractive than HPLC • Pharmacia’s BioPilot and BioProcess systems are commercial FPLC systems designed for pilot and industrial scale use • Flowrates up to 400 L/h are achievable in BioProcess system

  39. Expanded bed chromatography-1 • Particulate matter in protein sample should be removed before conventional purification procedures • Expanded bed chromatography aims to overcome this requirement • Duration and cost decrease • Design considerations: • Bead density • Flow rate of mobile phase • Bead size distribution

  40. Expanded bed chromatography-2 • The use of beads with an appropriate diameter range is important for the generation of a stable expanded bed (100-300 μm)

  41. Purification of recombinant proteins • Same techniques but generally more straight forward because of high expression of recombinant protein • Specific peptide or protein tags can be incorporated for rapid purification • Polyarginine or polylysine tag: cation exchange chromatography • Polyhistidine tag: metal chelate chromatography • Flag (a synthetic peptide) tag: immunoaffinity chromatography • Removal of the tag is generally desirable afterwards

  42. Protein Inactivation and Stabilization

  43. Approaches to protein stabilization • Buffered solution • Temperature control • Minimization of processing time • Avoid vigorous agitation or addition of denaturing chemicals • Add substances inactivating known inactivators • Include stabilizing agents • Glycerol, sugars and PEG: they decrease free water activity • BSA: as “bulking” protein

  44. Storage Optimization of storage conditions is a trial and error process... • Optimum T and pH for maximum stability • In liquid format: add stabilizing agents, filter-sterilization is advised • In frozen format: quickly freeze the solution, preferably in liquid nitrogen, then store in -70OC • In dry format: protein may be more stable

  45. Lyophilization-1 • Lyophilization involves the drying of protein directly from frozen state • Freeze the sample • Apply vacuum • Increase the temperature  sublimation • Many commercial proteins (e.g. vaccines, hormones, antibodies) are marketed in freeze-dried form

  46. Lyophilization-2 • One of the least harsh method for protein drying • Lightweight product  distribution easier • Can be rapidly rehydrated • Accepted by regulatory authorities • Equipment is extremely expensive • Running cost high • Long processing times • Some proteins exhibit an irreversible decrease in biological activity

  47. Characterization-1

  48. Characterization-2 • Functional Studies • Determination of specific activity • Determination of substrate range and specifity • Kinetic characteristics • Effect of various influences on activity

  49. Characterization-3 • Evidence of purity 1-D SDS-PAGE: The most common method used is 1-D polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS) Purpose: • Determination of purity • Determination of molecular mass

  50. Characterization-4

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