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Introduction to protein purification IGP methodology 20052006

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Why does the protein of interest need to be purified? What is the source ... Gradient elution not possible (would require stepwise change) Column Chromatography ... – PowerPoint PPT presentation

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Title: Introduction to protein purification IGP methodology 20052006


1
Introduction to protein purificationIGP
methodology 2005-2006
  • Pranav Danthi
  • PostDoc/Dermody lab
  • pranav.danthi_at_vanderbilt.edu

2
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?

3
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

4
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

5
Properties of protein affect purification strategy
6
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

7
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

8
Quantification of purification
9
Monitoring protein purification
10
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

11
Typical setup of FPLC
sample
pump
Buffer A
Buffer B
12
FPLC instrumentation
13
Protein purification is a multi-step process
14
Preparation, Extraction and Clarification
15
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

16
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17
Salting out of proteins using ammonium sulfate
18
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

19
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
20
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

21
Example of a tagging vector
22
Chromatogram from Ni affinity purification
23
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

24
Tandem Affinity Purification can be used to
identify interacting proteins
25
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26
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

27
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
28
  • 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?
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