Advanced protein purification IGP methodology 20052006

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Advanced protein purificationIGP methodology
2005-2006

• Pranav Danthi
• PostDoc/Dermody lab
• pranav.danthi_at_vanderbilt.edu

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Protein purification is a multi-step process
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Recap

• Set the aims (purity and quantity)
• Characterize the target protein
• Develop assay methods
• Select techniques and conditions compatible with
  sample stability.
• Use combinations of different separation
  principles.
• Start with high selectivity increase
  efficiency.
• Use few steps.
• Limit sample handling between purification step

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Types of purificationTake advantage of
biophysical characteristics

• Selective Precipitation/ Capture (based on
  solubility/ specific affinity)
• Capture/Intermediate
• Gradient Chromatography (based on charge
  properties)
• Capture/Intermediate/Polishing
• Size-exclusion Chromatography (based on size or
  shape)
• Intermediate/Polishing

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Gradient Chromatography

• Proteins stick selectively to a resin in an
  initial buffer
• Many proteins remain in the mobile phase and are
  discarded
• Elute the protein of interest by gradually
  changing the buffer to alter protein-resin
  interaction.

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Gradient Chromatographyexamples

• Ion exchange proteins stick in low saltelute
  as salt concentration increases
• eg. Mono S (cation exchange)
• eg. Mono Q (anion exchange)
• Hydrophobic interaction proteins stick in high
  saltelute as salt concentration decreases
• eg. phenyl-sepharose
• Reverse-phase Interactions are regulating by
  changing the polarity of the solvent they are in.
• eg. HPLC/C-18

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Ion exchange chromatography
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Ion exchange chromatography set-up
(Sample volume can be large)
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Typical elution profile
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Fractions 4-9 Pooled 23 U/mg Purification 2.3
fold
Specific Activity 10 2 f 12 28
50 25 16 8 U/mg
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Ion Exchange

• Anion vs. cation exchange
• Determine empirically
• Strength of the exchanger
• Strong (eg. Q anion or S cation)functional
  groups are strongly ionic your protein is more
  likely to stick, but so are other proteins (low
  stringency)
• Weak (eg. DEAE anion or carboxymethyl (CM)
  cation)if your protein sticks well, a higher
  fold purification is likely (high stringency)

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Hydrophobic interaction chromatography

• Stationary phase Non-polar (octyl or phenyl)
  groups attached to
• an inert matrix
• Resembles salting out
• Load column at high ionic strength,
• water of hydration is removed from
  proteins
• Those with fewer polar residues on the surface
  lose water easiest
• Possible elution strategies
• Decreasing salt concentration (since higher salt
  augments hydrophobic interactions)
• Increasing concentrations of detergent
• Changes in pH

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GF Separation based on size

• Very large proteins (or protein complexes or
  other molecules) traverse the column quickly
  elute first
• Very small proteins have the largest accessible
  volume and the farthest to travel traverse the
  column very slowly elute last
• Proteins/complexes between these extremes
  traverse the column at varying speeds

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Gel Filtration/Size Exclusion
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Gel Filtration/Size Exclusion
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Choice of a size exclusion resinExample
Pharmacia
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GF Separation based on size
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(No Transcript)
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Size exclusion can be used to determine apparent
molecular weight

• Retention time of protein is inversely
  proportional to apparent molecular weight
• Calculate Kav based on elution volume of protein
  on the column and the void volume of column
  Kav (Ve-Vo)/(Vt-Vo)

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Summary of chromatographic methods

• Method Capacity Position
  Expense
• Ion-exchange high early
  low
• Hydrophobic high early mid
• Gel-filtration low late (last) low
• Affinity high any (only?) mid

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Remember

• There is no magical protocol for protein
  purification.
• Need to carefully optimize the method and
  strategy for each protein.
• The more you know about your protein the better
  you can design the purification strategy.

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Problems

• What may be another way of eluting proteins that
  are bound on an ion exchange resin besides using
  a salt gradient? How would this work?
• You have 2 purified proteins Protein X has a
  molecular weight of 15KDa and forms a trimer,
  protein Y has a molecular weight of 26KDa and
  forms a dimer. How would you determine if X binds
  Y. How would you determine the stoichiometry of
  interaction if they do bind?
• You have managed to precipitate your protein of
  interest using 40 ammonium sulfate. To remove
  the remaining impurities, you choose to use
  cation exchange chromatography. However, your
  protein fails to bind to the resin and is found
  in the flow through. What are some possible
  reasons for this?