Protein Purification Methods

Protein Purification Methods

1. Gel Filtration Chromatography

   • Load mixed proteins into gel filtration matrix of porous beads
     • Sepearation by M.W
       • Large proteins elute first
   • Useful features of gel filtration chromatography:
     • separation by M.W
     • beads are relatively inexpensive
     • beads come in a variety of pore sizes
   • Disadvantages of gel filtration chromatography
     • sample must be concentrated to a small volume before loading to the column
     • relatively low capacity- not good method for large-scale purification
     • resolution of similar-size proteins is poor
     • sample becomes more diluted during purification

2. Ion Exchange Chromatography

   • Anion exchange
     • Use positively charged beads to bind proteins with negative charges on their surface
     • Proteins are eluted with NaCl solution of increasing concentration, where Cl- ions displace protein from binding sith
       • Proteins are separated based on what conc. of Cl- is required to release them from the anion exchange bead
     • "+" groups in anion exchange bead are typically amino groups
     • Strong and weak ion exchangers
       • Strong ion exchangers: little pH dependence
         • Ex: Quaternary amine agarose
       • Weak ion exchangers: Functional group is a weak acid. Exhibits some pH dependence
         • Ex: Diethylamino ethyl agarose
   • Cation exchange
     • Strong and weak ion exchangers
       • Weak ion exchangers: Functional group is a weak acid. Exhibits some pH dependence
         • ex: carboxymethyl agarose
       • Strong ion exchangers: little pH dependence
         • ex: sulfopropyl agarose
     • positive charges on proteins bind to negative charges on beads
     • Examples of common cation exchangers: carboxymethyl agarose and sulfopropyl agarose
   • ion exchange beads are typically made of agarose
   • Load mixed proteins into ion exchange matrix
     • proteins are separated based on their ability to bind to the ion exchange matrix
   • How can proteins be eluted?
     • pH change (not common)
     • salt concentration "step" (commonly used, not recommended)
     • salt concentration "gradient" (recommended)

3. Hydrophobic Chromatography

   • Column matrix typically has a hydrophobic group covalently attached to agarose beads
   • Proteins bind at high salt concentrations, elute by decreasing salt concentration
   • Ex: Octyl agarose or Phenyl agarose

4. Affinity Chromatography

   • IMAC
     • "Immobilized Metal Affinity Chromatography"
     • used in purifying histidine tagged proteins
       • Thrombin is often used to remove the histidine tag
         • Thrombin cleavage will result in a protein with an extra Gly-Ser
     • elute using histidine or imidazole
       • Dialysis is a common way to get rid of unwanted small molecules, such as imidazole