Protein Purification Hjp

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Protein Purification Hjp

  1. 1. Protein Purification
  2. 2. Levels of Structure in Protein • Primary: A description of all covalent bonds. The sequence of AA residues • Secondary: particularly stable arrangements of AA giving rise to recurring structural patterns. • Tertiary: All aspects of the 3D folding of a polypeptide. • Quaternary: The spatial arrangement of multisubunits protein
  3. 3. Protein Purification • Crude extract: breaking cells, by osmosis lysis or homogenization. • Fractionation: separate proteins into different fraction based on size of charge. • Salting out: The solubility of proteins is lowered at high salt concentration. Ammonium sulfate ((NH4)2SO4). • Dialysis is a procedure to separate proteins from solvents
  4. 4. Guidelines for protein purification • Define objectives • Define properties of target protein and critical contaminants • Minimize the number of steps • Use a different technique at each step • Develop analytical assays Adapted from: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition AC
  5. 5. How pure should my protein be? Application Required Purity Therapeutic use, in vivo Extremely high > 99% studies Biochemical assays, X-ray High 95-99% crystallography N-terminal sequencing, antigen for antibody Moderately high < 95% production, NMR
  6. 6. Separation of proteins based on physical and chemical properties • Solubility • Binding interactions • Surface-exposed hydrophobic residues • Charged surface residues • Isoelectric Point • Size and shape
  7. 7. Basic scheme of protein purification From: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition AC
  8. 8. Protein preparation, extraction, clarification Cell growth, protein over- expression Cell lysis Removal of cell debris
  9. 9. Expression of Target Protein in E. coli Plasmid with dinI DinI transformation expression E. coli
  10. 10. Protein isolation, concentration, and stabilization Reversible precipitation with salt or organic molecules
  11. 11. Fractional precipitation of proteins Discard pellet Precipitate contaminants Add Add Precipitant, Centrifugation, Precipitant, Chromatography Discard supernatant, Centrifugation Resuspend protein Precipitate Discard protein of supernatant, interest Resuspend protein
  12. 12. Intermediate Purification Liquid chromatography (lower resolution, lower cost)
  13. 13. Types of liquid chromatography Adsorption Chromatography – Proteins bind to stationary phase – Proteins eluted by altering mobile phase – Includes: affinity, hydrophobic interaction, ion exchange, and chromatofocusing Solution Phase Chromatography – Proteins do not bind to stationary phase – Progress of proteins through column impeded by matrix of stationary phase – Includes: size exclusion chromatography (aka gel filtration)
  14. 14. Types of liquid chromatography Adsorption Resin Chemical Equilibrate Elution Type Separation By Names of Resins or Solution Group With With Metal, Ig, Low High Hydroxyapatite, Affinity Adsorption Specific Ligand Ligand Binding [Ligand] [Ligand] Heparin Sepharose, Any ligand Butyl sepharose, Octyl Hydrophobic Hydrophobic Hydrophobic Adsorption High Salt Low Salt sepharose, Phenyl Interaction Groups Effect sepharose Positively charged Coulombic Mono-Q, Source-Q, Anion Exchange Adsorption Low Salt High Salt ions Interacions DEAE Negatively charged Coulombic Mono-S, Source-S, Cation Exchange Adsorption Low Salt High Salt ions Interacions CM Negatively charged pH Chromatofocusing Adsorption Isoelectric Point Poly-buffer Mono-P ions gradient Size Exclusion Solution Size / Shape of Sephacryl #, Pores Same Buffer (gel filtration) Phase Protein Sephadex #
  15. 15. Polishing steps Liquid chromatography (higher resolution, higher cost) From: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition AC
  16. 16. Liquid chromatography techniques advantages and disadvantages Type of Advantages Disadvantages Resolution Chromatography Resins and ligands Affinity Quick and specific can be expensive Low to Medium Can be used directly Relatively low Hydrophobic Interaction from ammonium resolution and binding Low to Medium sulfate precipitation capacity Protein solution must Ion Exchange Versatile resin choices start at low [salt] Medium to High pH gradient can be Chromatofocusing High resolution harsh for protein High Distinct from other techniques, Can be Size Exclusion used analytically or for Long run time Low to High buffer exchange
  17. 17. Protein detection methods • SDS-PAGE – Visual confirmation • UV Spectrophotometry – Absorbance @ 280 nm – Due mostly to Trp – [Protein] calculated with Beer’s Law • Colorimetric Techniques – Color change proportional to [protein] – Bradford, Lowry, BCA J.S.C. Olson and John Markwell. Current Protocols in Protein Science (2007) 3.4.1-3.4.29
  18. 18. Final steps in purification • Check purity by detection methods • Test for interfering contaminants – Nucleases – Proteases – Toxins • Concentrate your protein – Precipitation – Centricons – Small column with high binding capacity • Choose a storage buffer and storage conditions – Consider intended use of protein – Stabilizing additives – Flash freeze protein and store at -80o C • Confirm identity of purified protein – Mass spectrometry – N-terminal sequencing – Analytical assays
  19. 19. Basic scheme of protein purification Cell growth, Liquid protein over- chromatography expression Reversible (lower resolution, precipitation lower cost) Cell lysis with salt or Removal of organic cell debris molecules Liquid chromatography (higher resolution, higher cost)
  20. 20. Separation Processes that can be Used to Fractionate Proteins Separation Process Basis of Separation Precipitation ammonium sulfate solubility polyethyleneimine (PEI) charge, size isoelectric solubility, pI Chromatography gel filtration (SEC) size, shape ion exchange (IEX) charge, charge distribution hydrophobic interaction(HIC) hydrophobicity DNA affinity DNA binding site immunoaffinity (IAC) specific epitope chromatofocusing pI Electrophoresis gel electrophoresis (PAGE) charge, size, shape isoelectric focusing (IEF) pI Centrifugation sucrose gradient size shape, density Ultrafiltration ultrafiltration (UF) size, shape
  21. 21. Protein Purification: Column Chromatography • The expansion of the protein band in the mobile phase is caused by separation of proteins with different properties and by diffusional spreading. As the length of the column increases, the resolution of two types of protein improves. • Rate is decreased and resolution can decline because of the diffusional spreading
  22. 22. Ion-exchange Chromatography • Cation exchangers contain negatively charged polymer • Anion exchangers contain positively charged polymer. • Is effected by pH
  23. 23. Size-Exclusion Chromatography • Also called gel filtration: The column matrix is a cross- linked polymer with pores of selected size. • Larger protein migrate faster than smaller ones because they are too large to enter the pores
  24. 24. Affinity Chromatography • Separate protein by their binding specificities. The proteins retained on the column are those that bind specifically to a ligand cross- linked to the beads. Proteins that do not binds to ligands are washed through to column
  25. 25. Electrophoresis • Separation of porteins is based on the migration of charged protein in an electric field • The migration of a protein in a gel during electrophoresis is a function of its size and shape µ = V/E = Z/ f µ : electrophoretic mobility V: velocity; E: electrical potential Z: net charge; f: frictional coefficient
  26. 26. SDS-PAGE: Sodium Dodecyl Sulfate (SDS) Polyacrylamide Gel Electrophoresis • SDS binds to most proteins probably by hydrophobic interaction. One SDS for every two AAs, Thus, each protein has a similar charge- to-mass ratio. • Coomassie blue stains protein. Western blot
  27. 27. Estimating the Molecular Weight of a Protein
  28. 28. Isoelectric Focusing • pI of a protein: net charge=0 • A pH gradient is established by allowing a mixture of organic acids and bases (ampholytes). Protein migrates until it reaches the pH that matches its pI
  29. 29. Two-Dimensional Electrophoresis • Separates proteins of identical MW that differ in pI or proteins with similar pI but different MW.
  30. 30. Activity Vs. Specific Activity • Unit: amount of enzyme causing transformation of 1 µ mole of substrate per min. at 25 oC under optimal conditions • Activity: Total units of enzyme (U). • Specific activity: (U/mg) of total protein
  31. 31. Bacterial expression vectors
  32. 32. Bacterial expression vectors
  33. 33. Mammalian expression vector

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