Protein Chromatography

• Chromatography


  A technique for analyzing or separating mixtures of gases, liquids or dissolved substances

  
• Chromatography


  • Involves two distinct phases: stationary phase (matrix) and mobile phase

  
• First records of chromatography

  Mid 1800s
• Chromatography (as a term)

  Colour writing - separation of plant pigments - chlorophyll, carotenoids coined in 1900s
• Chromatography uses


  • Forensics - blood, arson, post mortem
  • Food regulation/testing - horsemeat scandal, nutritional information
  • Athlete testing - including horses! (combined liquid chromatography and MS)
  • Quality control - alcohol, analysis of when a food spoils, water samples, contaminents
  • Pharmaceutical industry - purification of antibodies, creating vaccines, purity of preparations
• Principles of chromatography


  • Mobile phase passed over stationary phase
  • Sample - mobile phase
  • Relative affinity for stationary phase allows for differentiation of sample

  
• Types of chromatography


  • Gas Chromatography (GC)
  • Liquid Chromatography (LC)
  • High-Performance Liquid Chromatography (HPLC)
  • Thin-Layer Chromatography (TLC)
  • Other chromatography techniques (exchange chromatography, affinity chromatography)

  
• Column chromatography


  • Separation of mixtures
  • Purification
  • Isolation of active components
  • Estimation of drugs in a formulation
  • Isolation of active constituents
  • Separation of diastereomers
• Column chromatography process


  1. Column equilibration

  2. Sample loading

  3. Elution

  4. Column cleaning
• Principle of column chromatography


  Adsorption - mobile phase is liquid, adsorbants - Si, Al, CaCO3, starch

  
• How to succeed in column chromatography

  • Stationary phase
  • Mobile phase
  • Removal of impurities
  • Affinity differences
  • Quantity of adsorbent used
• Column chromatography variables


  • Dimension of the column
  • Particle size of column packing
  • Activity of the adsorbent
  • Temperature of the column
  • Packing of the column
  • Quality of solvents

  
• Protein purification


  • Preparative vs. Analytical
  • Source
  • Preliminary steps
  • Purification strategies
  • Chromatography
  • Protein concentration
  • Yield & analysis
• Protein extraction


  1. Homogenization

  2. Sonication

  3. Freeze-thaw cycles

  4. Organic solvents
• Protein purification - preliminary steps

  • Precipitation and differential solubilization - salting out, detergents
  • Ultracentrifugation - sub-cellular organelles
• Need a pure preparation of a protein to determine properties or activity
• Protein chromatography techniques


  • Size-exclusion chromatography
  • Ion-exchange chromatography
  • Affinity chromatography
  • Hydrophobic interaction chromatography
  • Chromatofocussing
• Ion exchange chromatography


  • Anion-exchange chromatography
  • Cation-exchange chromatography
• At the pH of the mobile phase, peptide A has a pI of 5.1 and a net negative charge


  Peptide B has a pI of 7.8 and more positively charged residues at neutral pH
• Size exclusion chromatography


  Separates molecules based on differences in size as they pass through a porous stationary phase
• Affinity chromatography


  Based on binding affinity - beads in the column have a covalently attached ligand, any protein with affinity for the ligand binds to the beads
• Bound proteins eluted by a solution containing either a high concentration of salt or a free ligand
• Affinity chromatography


  Based on binding affinity between a ligand covalently attached to beads in a column and a protein with affinity for that ligand
• Affinity chromatography


  1. Beads in the column have a covalently attached ligand

  2. Any protein with affinity for the ligand binds to the beads, migration is retarded

  3. Proteins that do not bind flow more rapidly through the column

  4. Bound proteins eluted by a solution containing either a high concentration of salt or a free ligand that competes with the ligand attached to the beads, releasing the protein from the matrix

  5. Protein product that elutes from the column is often bound to the ligand used to elute it
• Hydrophobic interaction chromatography (HIC)


  Binding dependent on the surface hydrophobicity of the protein, binding enhanced by high ionic strength, [salt] concentration is lowered gradually and sample components elute in order of hydrophobicity
• Many proteins do not bind a ligand that can be immobilized on a column
• Genetic engineering for protein purification

  Gene encoding the target protein is fused to a gene encoding a peptide or protein that binds a simple, stable ligand with high affinity and specificity - the tag, tag sequences can be at amino or carboxyl terminus
• Genetic engineering for protein purification

  • GST tag - GST enzyme binds to glutathione immobilised on beads of agarose, target protein can be retrieved by washing with high concentration of salt or free glutathione to compete with immobilized ligand for GST binding, possible to remove the tag by protease cleavage
• Protein characterisation


  • Electrophoresis - polyacrylamide gel as a molecular "sieve" slowing migration of proteins in proportion to charge to mass ratio, SDS-PAGE for estimating purity and molecular weight, isoelectric focusing to determine isoelectric point (pI)
• Chromatofocusing


  Chromatographic medium equilibrated with a start buffer at a pH slightly above the highest pH required, sample applied with start buffer, elution buffer passed through to titrate the amines on the medium and the proteins - a gradient pH develops, proteins above their pI are negatively charged and retained near the top, those below their pI begin to migrate down and bind where the pH is above their pI
• 2D gel electrophoresis


  Combines SDS-PAGE and isoelectric focusing, more sensitive than either alone, separates proteins of identical molecular weight that differ in pI, or proteins with similar pI values but different molecular weight
• Post-chromatography processing


  1. Desalting - dialysis, desalting columns

  2. Concentration - lyophilization, ultrafiltration, chromatographic concentration, precipitation

  3. Yield and analysis - enzyme assay, protein assay, SDS-PAGE, Western blotting
• Recombinant proteins are a manipulated form of native proteins, generated in various ways to increase production, modify gene sequences, and manufacture useful commercial products
• Uses of recombinant proteins


  • Lab techniques - ELISA, Western blot, immunohistochemistry, enzyme assays, studying cellular responses
  • Therapeutic uses - hormones, interferons, interleukins, growth factors, blood clotting factors, enzymes for animal feed, lactic acid bacteria for food fermentation and nutrition
• Advantages of recombinant proteins

  • Ethical considerations, quick, cost-effective, scalable
• Disadvantages of recombinant proteins


  • Contamination, inactive protein, small proteins only, lack of post-translational modifications