Introduction to protein purification IGP methodology 20052006

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Introduction to protein purificationIGP
methodology 2005-2006

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

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Important questions to ask before you embark

• Why does the protein of interest need to be
  purified?
• What is the source of the protein?
• What is known about the protein?

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Why are proteins purified?

• High resolution structure or therapeutic use?
• gt99 purity is required
• Antibody generation?
• High yield of 90-95 pure protein
• Biochemical assays?
• Degree of yield/purity varies with application

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Sources of protein

• Non-engineered
• Examples Organ tissues or Cell lines
• The sequence of the protein may be unknown and
  purification is based on the activity of the
  protein
• Abundance of target protein is low

• Engineered
• Examples Expressed in Bacteria,
• Yeast, Insect or mammalian cell lines
• (transfected or transduced)
• The gene sequence for the protein is known and
  the gene for the protein is cloned
• Relatively higher abundance of protein
• Over-expressed proteins can accumulate in
  inclusion bodies

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Properties of protein affect purification strategy
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Quantification of purification

• Yield
• How much protein of interest is in the
  fraction?
• Amount of protein of interest

• Purity (Specific activity)
• What fraction of purified protein is the protein
  of interest?
• Protein of interest/total protein

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Measuring proteins

• Protein of Interest
• Activity assay
• - Enzyme assay
• - Bioassay
• Western blot (quantitative)
• - Need specific antibodies and standard curve

• Total protein
• Absorbance at 280nm
• - non-destructive
• Colorimetric assays
• Color is directly proportional to protein
  concentration
• Reaction is destructive
• Reaction is sensitive to buffer conditions

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Quantification of purification
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Monitoring protein purification
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Purification method

• Batch method
• - good for small scale purification
• quick and dirty
• Gradient elution not possible (would require
  stepwise change)

• Column Chromatography
• Large scale purification possible
• Columns give better resolution
• Can be automated using pump or FPLC

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Typical setup of FPLC
sample
pump
Buffer A
Buffer B
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FPLC instrumentation
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Protein purification is a multi-step process
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Preparation, Extraction and Clarification
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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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Salting out of proteins using ammonium sulfate
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Selective capture using affinity chromatography

• Enzyme Substrate, inhibitor, cofactor
• Antibody Antigen, virus, cell
• Lectin glycoprotein, cell receptor
• Nucleic Acids, Heparin histone, polymerases
• Hormone, vitamin Receptor, carriers

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Affinity Chromatography
Make or purchase affinity matrix Bind protein to
affinity matrix (sample volume can be
large) Wash extensively Elute by changing
conditions or providing soluble competitor
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Affinity chromatography of recombinant proteins

• Clone the gene for the protein of interest
  upstream or downstream of tag (make sure it is in
  frame!)
• Plasmids with tags and convenient restriction
  sites are commercially available
• GST Glutathione
• His Cobalt/Nickel
• MBP Amylose
• Fc Protein A
• Tag amino acids can be removed by engineered
  protease cleavage sites

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Example of a tagging vector
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Chromatogram from Ni affinity purification
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Protein over-expression may lead to aggregate
formation

• A large amount of over-expressed protein is found
  in the insoluble fraction
• Can modify conditions of induction or growth to
  reduce inclusion body formation
• Can isolate protein from insoluble fraction using
  denaturing conditions
• Proteins may then be refolded if desired after
  purification

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Tandem Affinity Purification can be used to
identify interacting proteins
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Summary

• Before you start
• Set the aims (purity and quantity)
• Characterize the target protein
• Develop assay methods
• Protocol development checklist
• 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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Problems

• You are purifying a his-tagged protein from E.
  Coli. lysate (lane L). The material from two
  different purifications are shown. Which (sample
  A or B) is purer? Why do you think so? If the
  material eluted looked like C what changes would
  you make to your affinity purification conditions
  to make it as pure as A or B?

B
L
A
C
Protein of interest
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• Is a purification technique that gives you a 60
  yield but only a 3-fold purification better than
  one that gives you a 10-fold purification but
  only a 25 yield?
• You are purifying a protein and you consistently
  see a band that reacts with an antibody specific
  to your protein of interest but is of much
  smaller size, what may the possible reasons for
  this?