Chromatography

Chromatography

1. Uses

   • Basic research
     • Studying individual proteins teaches us about general form and function
   • Pharmaceutical industry
     • The protein itself may be used as a therapy
       • Insulin
       • Interferon
       • Herceptin
     • Used in the development of small molecule inhibitors
       • Olaparib
       • Abiraterone

2. Underlying principles

   • Two distinct phases
     • Stationary phase
       • Immobile
     • Mobile phase
       • Flows freely over the stationary phase
   • Column chromatography
     • Separation
       • Partition
         • Between two liquid phases
       • Adsorption
         • To a solid phase
     • Components
       • Column
         • Glass, plastic or stainless steel
       • Stationary phase
         • Usually porous beads
           • 10-100um diameter
         • Can be matrix or resin
       • Mobile phase
         • Liquid or solution
         • Passed or pumped through stationary phase
         • Typically in buffer or solvent
   • Basic method
     • Solvent continuously pumped through column
     • Sample containing POI injected
     • Separated components are detected as they emerge from the column
       • Fractions collected
     • A chromatogram is continuously recorded
       • Typically UV absorbance
         • Tryptophan 280nm

3. FPLC

   • FAST PROTEIN LIQUID CHROMATOGRAPHY
     • Typical commercial chromatography equipment for protein purification
   • Method
     • Sample with POI applied in band to the top of the column
       • Components have different interactions with the stationary phase
         • Pass through the column at different rates
           • Separation
   • Resolution (separation) generally correlated with column length
     • HOWEVER component bands broaden or spread as they travel down
       • Diffusion/resin effects
   • Equipment
     • Stationary phase
       • Chromatography media
         • Spherical beads
           • Defined size
           • Porous
           • Proteins diffuse in and out
           • Can be chemically modified
           • Sephadex
             • Separation pharmacia dextran
             • Dextran covalently attached to highly cross-linked agarose
               • Dextran
                 • Complex polysaccharide
                 • Multiple glucose molecules
                   • Linear links
                     • 1-6
                   • Branches
                     • 1-3
                 • 3D network formed by additional chemical cross-linking
                   • Porous bead
                   • Can add additional chemical groups to the matrix
           • Sepharose
             • Separation pharmacia agarose
             • Highly cross-linked agarose
           • Sephacryl
             • Separation pharmacia bisacrylamide
             • Cross-linked allyl dextran and N,N'-methylene bisacrylamide
           • Changing properties
             • Cross-linking
               • Bead porosity
             • Chemical groups
               • Separation properties
         • Resin
         • Matrix

4. Protein properties

   • Affecting purification
     • Size
       • Shape
     • Charge
       • Positive
       • Negative
     • Solubility
       • Function of salt
       • pH
       • Temperature
     • Ligands
       • DNA
       • Peptide
       • Co-factors
   • Uses
     • Different types of chromatography

5. Size exclusion chromatography

   • Gel filtration
   • Separates globular proteins
     • According to molecular mass
   • No direct interaction between POI and stationary phase
   • Isocratic
     • "Equality of strength or power"
     • Single solvent
       • Buffer
       • Does not change composition throughout the experiment
   • Stationary phase
     • Porous beads
       • ~100um diameter
       • Various pore sizes
         • Large proteins
           • Don't fit into any pores
           • Pass around outside of beads
           • Carried with bulk flow of solvent
           • Elute first
         • Small proteins
           • Diffuse freely into and out of all pores
             • Shielded from bulk flow of solvent
           • Retained/retarded
           • Elute last
         • Medium proteins
           • Can enter large pores
           • Can't enter small pores
           • Somewhat carried along with bulk flow
           • Intermediate elution
   • Chromatograph
     • Elution profile
     • Column volume
       • Physical volume of the column, ml
         • (pi) x r(squared) x h
     • Vold volume V0
       • The volume that a very large protein sees
       • Volume (ml) that passes through the column before an unretained substance emerges at the detector
         • Physical volume of the column minus packed resin (bed) volume
           • SO this does not include the volume inside the beads!
         • Large proteins/molecules which do not fit inside the largest pore elute at void volume
     • Total volume Vt
       • The volume that a very small protein sees
       • The physical volume of the column plus the pore volume within the beads, minus the packed resin (bed) volume
       • Very small proteins which fit into all pores elute here
   • Intermediate sized proteins are separated from each other roughly according to size
   • All proteins/molecules elute within one column volume
   • Ideal resolution
     • Clear and even peaks
   • Practical use
     • Have to select media/column dependent on predicted/known size and stoichiometry of POI
       • Ideally POI properties need to fit in the middle of the range
       • Is it dimeric?
     • Can estimate the molecular mass if not known
       • Calibrate the column with standards
         • Blue dextran 2mDa
         • Bovine serum albumin 66kDa
         • Carbonic anhydrase 29kDa
         • Cytochrome C 14.2kDa
         • Aprotinin 6.5kDa
         • Calibration curve
           • Elution volume vs. Log10 Mr
             • Fit a line to the linear region
               • Exclude the proteins that elute at VO and Vt because they are not separated by the column.
             • The Mr can be determined by taking the antilog of the y-intercept value
     • Assumptions
       • Unknown protein has same shape as molecular mass standards.
         • Rod-shaped/cylindrical proteins have higher apparent molecular mass
           • Effective radius in solution increased relative to true Mr
       • Protein does not interact with resin
         • Those that interact are retained for longer and elute later
           • Mr underestimated
       • The protein is monomeric
         • May form oligomers
           • Elutes earlier
             • Molecular mass appears higher

6. Ion exchange chromatography

   • Separation roughly according to surface charge
   • POI interacts directly with surface of stationary phase
   • Isoelectric point pI
     • Basic amino acid residues lysine, arginine and histidine are positively charged on their amine group at pH below their sidechain pKa
     • Acidic amino acid residues aspartic acid, glutamic acid are only negatively charged at pH above their sidechain pKa
     • Proteins have different amino acid compositions and therefore different ratios of basic and acidic residues
       • Charge of protein depends on
         • Component amino acids
           • pKa
           • Chemical environment
         • pH of surroundings
     • Example: human Chk2
       • Neutral pH
         • Estimated charge -10
       • pH 8.5
         • Estimated charge -20
       • pI 6.0
     • pI: The pH at which a particular molecule carries no net electrical charge
   • Determining media
     • Experimental pH
       • = pI
         • No net charge
         • Does not bind to any ion exchange resin
       • <pI
         • Protein is positively charged
           • Binds negatively charged resin
             • Cation exchange resin
               • Sulphate
                 • Strong
                 • Charge maintained over pH range
               • Carboxylate
                 • Weak
                 • Charge maintained over limited range
       • >pI
         • Protein is negatively charged
           • Binds positively charged resin
           • Anion exchange resin
             • Quaternary amine
               • Strong
               • Maintain charge across pH range
             • Diethylaminoethane
               • Weak
               • Charge maintained over limited range
   • Principles
     • Reversible displacement of ions bound to a charged matrix
       • Matrix associated with counter-ions from buffer
         • Low strength <50mM NaCl
       • 1. Sample containing POI
         • Applied to matrix
           • Displaces counter-ions
         • 2. High ionic strength buffer applied
           • e.g. NaCl >1M
           • Competition for charged group on matrix
             • Displacement of bound POI
     • There is a linear gradient from low to high ionic strength which elutes the POI from the matrix
       • Created by mixing different proportions of buffer A with buffer B
   • Steps
     • 1. Equilibration
       • Prepare the chromatography column such that the buffer conditions are suitable to capture the POI
         • Low salt/ionic strength
     • 2. Sample application and wash
       • Apply the sample with the POI and wash with buffer to remove unbound components
         • Wash for about 5cv or until UV absorbance close to zero
     • 3. Elution
       • Elute bound proteins by applying a linear buffer gradient to the column
     • 4. Regeneration
       • Wash with high ionic strength bugger to remove remaining bound components

7. Affinity chromatography

   • Highly specific
     • Based on the interaction between POI and covalently modified resin
     • Many different types
     • Simplify purification
       • Genetically modify POI
         • Affinity tag
           • Polyhistidine
             • Immobilised metal affinity chromatography
               • Resin beads attached to immobilised metal ions
                 • Ni+ or Co+
                 • Link a nitriloacetic acid group to the resin by a spacer region
                   • Tetradendate chelation by acetate "arms"
                     • Leaves two of six interaction sites on ion free for interaction with polyhistidine tag
                       • Histidines in the affinity tag complete the coordination shell
                         • Removal
                           • Compete it off
                             • Competitive ligand
                               • Imidazole
                                 • Displaces polyhistidine tag from resin by competing for binding to nickel ion
             • 6-10 histidine residues fused to N or C terminus of POI
               • Rapid capture and purification by IMAC
               • 6 most common
             • Method
               • Similar to IEX
                 • Buffer for elution has 300mM imidazole
                   • Remove by washing with pH 5.0 buffer
                     • Protonates imidazole
                       • Can't bind to the metal
           • Strep-II
         • Fusion protein
           • Glutathione-S-transferase
             • Cytosolic protein
             • Cellular detoxification
               • Conjugation of glutathione (GSH) to wide range of alkylating agents
                 • Endogenous
                 • Xenobiotic
                 • Naturally occurring tripeptide
                   • Glu-Cys-Gly
                     • Unusual Glu-Cys linkage
             • Glutathione sepharose
               • Sepharose bead linked to glutathione by thiol group of cysteine residue
             • Fuse GST to N-terminus of POI
               • Clone the coding sequence into an expression vector
                 • pGEX series
               • Fusion protein expressed as monomer
                 • Rapidly dimerises
                   • Perhaps not suitable if POI dimerises/oligomerises
             • Purification
               • Very similar to IEX
                 • Buffer with 20mM glutathione
                   • Elute by "competing off" with the same/similar ligand
                   • Remove glutathione from column by alternate washes with pH8.5/pH4.5 buffers
           • Maltose binding protein
         • Take advantage of these properties

8. Purification in practice

   • Several sequential chromatography steps
     • To fully separate from other proteins in source material
     • Each step should increase POI purity and activity
       • Without decreasing yield
   • The strategy is tailored to the POI
   • Capture, immediate purification, purification
     • 2-4 steps
   • Example: Chk2 kinase domain
     • Expressed as a GST fusion protein in E. coli
     • 1. Host transformed with plasmid encoding recombinant protein
       • 2. Cell culture grown at 37c
         • 3. IPTG added to induce protein expression
           • 4. Cells harvested by centrifugation
             • 5. Cell pellet frozen
               • 6. SONICATION: Cell wall mechanically disrupted
                 • 7. Centrifugation to remove insoluble material
                   • Starting sample
                     • 8. Glutathione sepharose affinity chromatography
                       • 9. SEC
                         • 10. Affinity tag removal
                           • 11. Glutathione sepharose affinity chromatography
                             • Purified POI
                               • Structural/functional studies