Protein Purification bl4010 10. 19. 06 Resources

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Protein Purification

  • BL4010 10.19.06


  • 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  • Note that whatever book you get, it is already likely to be out of date.

Why purify?

  • in vitro vs. in vivo analysis

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

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

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

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

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

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


  • 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

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


  • 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)

Purification schemes

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.


  • Spectroscopy
    • A280 e 1%280 = 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


  • 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 pH>10
    • 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.

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

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.

Methods of concentration

  • Dialysis
  • Filtration

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

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?


  • 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)


  • 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).


  • 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

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

Ionic Exchange Chromatography

Hydrophobic interaction chromatography

  • Hydrophobic group bound to solid phase
  • Binding
  • Elution
    • decrease salt
    • add detergent
    • decrease polarity
    • of mobile phase

Affinity Chromatography

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


  • Tris-glycine buffer
  • 10% SDS



  • Protein detection
    • Coomassie blue
    • Sypro
    • Cybergreen
    • Silver staining
  • coomassie brilliant blue A595

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)

Using antibodies

Western blotting

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

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

Protein purification simulation


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


  • 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 pH>10
    • 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.

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

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

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

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