wk 6

• Why do we need to purify proteins?

  To separate the desired protein from the other proteins in the sample (e.g. from bacteria, yeast, cultured cells, or host organisms)
• What are we purifying the proteins from?

  • Bacteria (E.coli)
  • Yeast (Unicellular eukaryotes)
  • Cultured cells (Mammalian cell line (HeLa/CHO))
  • Host (Mouse/plant/organ)
• What makes a good source?

  • Quantity (Does it contain a large quantity of the desired protein?)
  • Quality (Is the protein going to be in the most suitable form?)
  • Ease (Are the cells easy to grow? Are the cells easy to open?)
• Your protein is in a soup of many others!
• Cell lysis


  The process of breaking open cells to release their contents
• Cell lysis


  1. Detergent/Solubilise membrane

  2. Mechanical

  3. Buffer

  4. Salts
• What happens if we get the conditions wrong during cell lysis?
• Why centrifugation?


  Particles with greater density sink (sediment), and the greater the difference in density, the faster they move. Applying centrifugal force makes this happen more quickly than when left to gravity alone.
• How do we purify proteins?

  • Size (Size exclusion chromatography, dialysis)
  • Charge (Anion exchange, Cation exchange)
  • Solubility (Centrifugation, sedimentation)
  • Affinity (Antibody, Affinity tags (recombinant e.g. His-tag))
• PAGE (Polyacrylamide gel electrophoresis)


  A technique used to measure the success of protein purification by separating proteins based on their size and charge
• SDS-PAGE


  Sodium dodecyl sulfate polyacrylamide gel electrophoresis, a denaturing gel electrophoresis technique that separates proteins based on their molecular weight
• Enzymes


  • Sensitive to pH, temperature, substrate concentration, salt concentration, type of buffer, and other chemicals
• Enzyme reaction rate = assay


  Measure rate of loss of substrate or gain of product
• Artificial substrate that is either coloured or fluorescent is often used to measure enzyme activity
• Decreasing substrate initial rate is used to determine the kinetic constants KM and Vmax for an enzyme
• Protein purification


  A practical approach
• In the lecture we will link the physical properties with suitable approaches for protein purification
• How do we purify a protein?

  1. Homogenise sample

  2. Lyse cells

  3. Clarification

  4. Purification

  5. Assessment of purity
• Homogenisation


  • Opening the cells
  • Whole cell
  • Cytoplasm is reducing
  • Lysed cell is oxidising
• Clarification


  • Soluble proteins in the supernatant
  • Insoluble proteins and membranes in the pellet
• Physical properties used for protein purification

  • Solubility
  • Charge
  • Affinity
  • Size
• Affinity chromatography


  Protein interacts with a bead or resin
• Binding affinity chromatography


  • His-tag protein binding to Ni beads
  • Elution with imidazole
• Ion-exchange chromatography


  • Charged proteins bind to the resin
  • Weakly positive proteins elute first
  • Increasing salt concentration elutes more positively charged proteins
• Dialysis


  • Small molecules transverse semi-permeable membrane
  • Larger molecules stay inside
• Size exclusion chromatography


  • Separate according to size
  • Accounts for native state of protein complex
  • Large molecules flow more rapidly through column
• Let's apply this in our next session
• Protein purification steps


  • Homogenise sample
  • Lyse cells
  • Centrifugation
  • Purification
  • Assessment of purity
• PAGE


  Polyacrylamide gel electrophoresis
• Polyacrylamide gel electrophoresis (PAGE)

  1. Velocity of migration depends on electric field, net charge on the protein, and frictional coefficient

  2. Frictional coefficient depends on mass of molecule, shape of molecule, and viscosity of the medium
• Native PAGE


  • Protein is separated in its folded state
  • Complex stays intact
  • Affected by net charge of protein
• SDS-PAGE


  • SDS = sodium dodecyl sulfate = detergent
  • Reducing agent
  • Denaturing gel
  • Separate according to molecular mass
  • Most common method for analysing purity
  • Pure sample - single band
• Staining methods


  • Coomassie
  • Silver stain
  • Ponceau S
• Isoelectric Focusing (IEF)


  • Separate according to pI (pI=pH where protein has no charge)
  • pH where net protein charge (z)=0, electrophoretic mobility (ν) =0
  • pH gradient gel
  • Proteins move until they reach pH=pI
• 2D gel electrophoresis


  1. Part 1 = IEF

  2. Part 2 = SDS-PAGE, run perpendicular
• Western blot


  1. Part 1 = SDS-PAGE

  2. Part 2 = transfer to membrane

  3. Part 3 = antibody specific to the protein
• Compare methods and when to use each of them and why