Protein Purification

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Protein Purification

• BL4010 10.19.06

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Resources

• Protein purification A practical approach.
  (Harris Angal IRL Press)
• Protein purification Design and scale-up of
  downstream processing. (Wheelwright Hanser Press)
  
• Methods in Enzymology - several volumes are
  concerned exclusively with protein purification.
• Note that whatever book you get, it is already
  likely to be out of date.

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Why purify?

• in vitro vs. in vivo analysis

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Why purify?

• By purifying a protein it can be clearly
  established that a particular biological activity
  (enzymatic activity, signaling capacity, etc.)
  actually resides in a unique protein.
• Purified proteins serve as extremely valuable
  biochemical reagents
• Determine mechanism (controlled, observable
  environment)
• Structural determination
• Sequence determination
• Antibody production
• Structure/function analysis - genetic engineering
• Finding inhibitors
• Detailed kinetic studies

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The basic techniques

• Concentration (size)
• precipitation
• ultrafiltration
• dialysis
• centrifugation
• Chromatography (size/charge/chemistry)
• ion exchange
• size exclusion
• affinity

• Electrophoresis (size/charge)
• "native"
• denaturing
• isoelectric focusing
• 2-dimensional
• Immunological
• (size/charge/chemistry)
• chromatography
• in situ imaging
• immunoblotting

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Getting started

• Assay (measurable quality) must be specific and
  convenient
• measuring a change in absorbency as NADPH is
  oxidized in a coupled reaction,
• binding activity
• a shift of a labeled molecule (DNA, protein) on a
  gel
• the transformation of substrate
• the ability to stimulate cell reaction (e.g.
  proliferation)
• Source
• easier to purify from a rich source vs. a poor
  source

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Protein Purification Principles

• Define objectives
• for purity, activity and quantity required of
  final product to avoid over or under developing a
  method
• Define properties of target protein and critical
  impurities
• to simplify technique selection and optimisation
• Develop analytical assays
• for fast detection of protein activity/recovery
  and to work efficiently
• Remove damaging contaminants early
• for example, proteases

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Protein Purification Principles

• Use a different technique at each step
• to take advantage of sample characteristics which
  can be used for separation (size, charge,
  hydrophobicity, ligand specificity)
• Minimize sample handling at every stage
• to avoid lengthy procedures which risk losing
  activity/reducing recovery
• Minimize use of additives
• additives may need to be removed in an extra
  purification step or may interfere with activity
  assays
• Minimize number of steps - KEEP IT SIMPLE!
• extra steps reduce yield and increase time,
  combine steps logically

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Starting materials

• Natural source or artificial expression system
• Host for expression,
• Bacteria, yeast, plants, transgenic animals
• Abundance, contaminants
• Lysis and clarification procedures
• Native or denaturing conditions
• Subcellular fractionation
• Selective precipitation
• PEI, Streptomycin Sulfate, CTAB for RNA/DNA
• Ammonium Sulfate for Proteins

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Capture

• Quickly remove most damaging contaminants
• Concentrate, adsorption methods
• Ion Exchange most general
• Affinity chromatography can combine capture,
  intermediate and polishing steps
• This step should remove most unwanted contaminants

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Intermediate purification

• Use a different technique
• Affinity chromatography, Hydrophobic interaction
  chromatography
• Starting conditions are specific for each
  technique
• Buffer must be compatible with adsorption
• Can change buffer by dialysis or desalting by GFC
• Adsorption techniques result in small volume
  concentrated sample

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Polishing

• Final removal of trace contaminants
• Often size exclusion chromatography
• Buffer exchange is a part of the process
• Sample volume always increases need to start with
  a concentrated sample
• Sample can be concentrated by
• Precipitation (selective or nonselective)
• Ultrafiltration (dialysis under pressure)

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Purification schemes
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Assays, Quantitation and Documentation

• Assay enzyme activity at every step
• Contaminants at early stages can mask or inhibit
  activity
• Inactivation can occur at high temperatures,
  because of proteolysis, oxidation, aggregation,
  etc.
• Assay total protein
• Run an SDS gel to visualize specific contaminants
• Specific activity is defined as units of
  enzymatic activity per unit of total protein -
• Yield can be defined in terms of total protein
  mass, and total enzyme units
• Goal is a high yield and high specific activity.

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Detection

• Spectroscopy
• A280 e 1280 14.5 g-1Lcm-1
• 10 mg/ml A280 14.5
• cofactors
• Protein Assay
• Bradford (coomassie)
• Biuret (copper)
• Lowry (modified biuret - phosphomolybdotungstate
  mixed acid reduced by Cu2 and F,Y,W to form
  heteropolymolybdenum blue A750
• Enzyme Assay

A550
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Assays

• Enzymatic assays
• PNPP is hydrolyzed to PNP and Pi
• Fixed time assay
• Mix enzyme and substrate, react for a fixed time,
  s
• top the reaction with a strong base,
• read the concentration of PNP at pHgt10
• Continuous assay
• Monitor PNP production directly in the spec at ph
  8
• Bradford Assays for total protein
• SDS page for the distribution of proteins by
  size.

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Assay and Specific Activity
Fraction Volume (ml) Total protein (mg) Total activity Specific Activity Percent Recovery Fold Purificat'n
Crude extract 3,800 22,800 2460 0.108 100 0
Salt ppt. 165 2,800 1190 0.425 48 3.9
IEC 65 100 720 7.2 29 66
SEC 40 14.5 555 38.3 23 355
Affinity 6 1.8 275 152 11 1407
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Criteria for purity

• When is protein pure or pure enough?
• Homogeneity
• protein complexes?
• Constant specific activity
• Practical further attempts at purification are
  futile since the only material left in the
  fraction is the material that actually is
  responsible for the activity being assayed.

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Methods of concentration

• Dialysis
• Filtration

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Protein Precipitation

• "Salting Out" when enough salt has been added,
  proteins precipitate
• cold prevents denaturation
• collect by filtration or centrifugation
• redissolved in solution using a buffer with low
  salt content.
• works best with divalent anions like sulfate,
  especially ammonium sulfate which is highly
  soluble at ice temperatures

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Buffer Exchanges

• Almost all purification steps will be a buffer
  with specific pH and/or ionic strength
• The buffer used impacts the protein's biophysical
  characteristics
• Why exchange?
• e.g. If you have just precipitated a protein with
  ammonium sulfate, you obviously now have that
  protein in a high salt environment.
• How can you remove salt?

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Centrifugation

• Zonal centrifugation Mixture to be separated is
  layered on top of a gradient (e.g. sucrose or
  ficoll) increasing concentration down the tube -
  can be continuous or discontinuous (layers)-
  provides gravitational stability as different
  species move down tube at different rates forming
  separate bands.
• Species are separated by differences in
  SEDIMENTATION COEFFICIENT (S)  Rate of movement
  down tube/Centrifugal force
• S is increased for particle of LARGER
  MASS(because sedimenting force a M(1-vr)
• S is also increased for MORE COMPACT STRUCTURES
  of equal particle mass (frictional coefficient is
  less)

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Centrifugation

• Isopycnic (equal density) centrifugation
  Molecules separated on EQUILIBRIUM POSITION, NOT
  by RATES of sedimentation.Each molecule floats
  or sinks to position where density equals density
  of solution (e.g. CsCl gradient for nucleic acid
  separation).

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Chromatography

• Chromatography a broad range of physical methods
  used to separate and or to analyze complex
  mixtures.
• The components to be separated are distributed
  between two phases a stationary phase bed and a
  mobile phase which percolates through the
  stationary bed.

paper chromatography stationary phase paper
mobile phase solvent
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Size-Exclusion Chromatography

• Separation of proteins based on kinetics of
  moving through the available space (larger
  proteins have less space than smaller molecules)
• Proteins larger than matrix elute in void volume
  (1 exchange of volume outside beads)
• Proteins smaller than matrix partition in and out
  of beads
• Pore size in beads is not uniform
• Also some surface interaction with beads

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Ionic Exchange Chromatography
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Hydrophobic interaction chromatography

• Hydrophobic group bound to solid phase
• Binding
• high salt (increases water surface tension,
  decreases available water molecules, increases
  hydrophobic interactions)
• Elution
• decrease salt
• add detergent
• decrease polarity
• of mobile phase

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Affinity Chromatography

• Ligand can be a small molecule, metal or antibody
• Protein binds specifically to ligand attached to
  matrix
• Elution with free ligand

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Electrophoresis

• Tris-glycine buffer
• 10 SDS

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Electrophoresis
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Electrophoresis

• Protein detection
• Coomassie blue
• Sypro
• Cybergreen
• Silver staining

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Using antibodies
Antibodies (immunoglobulins) bind specific
antigens/epitopes monoclonal - all bind same
epitope polyclonal - mixture that binds several
epitopes Secondary antibodies -
anit-immunoglobulins (antibodies to antibodies)
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Using antibodies
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Western blotting

• Separate proteins by electrophoresis
• Transfer to membrane (e.g. nitrocellulose)
• Bind primary antibody
• Bind secondary antibody
• Detection

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Immuno-Affinity Chromatography

• Antibody fixed to matrix
• Protein binds to antibody
• Wash unbound and loosely bound proteins off
  column
• Elute protein with change in salt/pH

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Protein purification simulation

• http//www.tlsu.leeds.ac.uk/courses/bioc2060/prote
  inlab102/proteinlab.html

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Example Purification of Alkaline Phosphatase (AP)

• Periplasmic Protein in E. coli
• The space between the rigid peptidoglycan cell
  wall and the osmotically sensitive plasma
  membrane
• Phosphate scavenger
• Liberates Pi from a variety of substrates
• Induced by phosphate starvation
• Used to remove terminal phosphates for selective
  DNA ligation reactions
• Heat stable, Zn enzyme

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Assays

• Enzymatic assays
• PNPP is hydrolyzed to PNP and Pi
• Fixed time assay
• Mix enzyme and substrate, react for a fixed time,
  s
• top the reaction with a strong base,
• read the concentration of PNP at pHgt10
• Continuous assay
• Monitor PNP production directly in the spec at ph
  8
• Bradford Assays for total protein
• SDS page for the distribution of proteins by
  size.

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Text Book Purification

• 1. Lysozyme treatment to release periplasmic
  proteins
• Centrifugation to separate soluble AP from cells
• Dialysis to remove starting buffer (overnight)
• 2. Heat treatment to precipitate weaker proteins
• Centrifugation to separate soluble AP from
  insoluble PPT
• Ammonium sulfate to concentrate proteins/remove
  non protein contaminants
• Dialysis to remove ammonium sulfate (O/N)
• 3. Anion exchange (DEAE) chromatography
• Step elution with 0.125M Salt
• 4. SDS Page to quantify the proteins in each
  fraction

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Starting material

• E. coli cells starved for phosphate
• Sucrose shrinks the plasma membrane reduces
  turgor pressure
• Lysozyme cleave glycosidic linkages in cell wall
• DNAse reduces viscosity from inadvertantly lysed
  cells
• Left with AP, DNAse, Lysozyme, Sucrose other
  periplasmic and cytoplasmic contaminants

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Alternative strategy

• Osmotic shock used to liberate periplasmic
  proteins
• Many fewer proteins in periplasm than cytoplasm
• Sucrose draws water from cytoplasm, shrinks inner
  membrane
• EDTA permeabilizes cell wall
• Transfer to low osmotic strength buffer causes
  the inner membrane to slam into the cell wall and
  force out periplasmic proteins
• Periplasmic proteins, no lysozyme, no DNAase, not
  much sucrose