Current Approaches to Protein Purification Richard Burgess University of WisconsinMadison

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Protein Purification (from a lecture by Dr.
Richard Burgess, University of Wisconsin,
Madison, at the CSH protein purification
course). Object to separate a particular protein
from all other proteins and cell components
There are many proteins (over 4300 genes in E.
coli) A given protein can be 0.001-20 of total
protein Other components nucleic acids,
carbohydrates, lipids, small molecules Enzymes
are found in different states and locations
soluble, insoluble, membrane bound, DNA bound,
in organelles, cytoplasmic, periplasmic,
nuclear
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Study Question You are given a shoe box full
of an assortment of small objects including Ping
Pong balls Sugar cubes Paper clips 1/2 brass
screws Iron filings 1. List the properties of
each of these components that might help you
fractionate them. 2. Devise the most efficient
method you can for getting pure paper clips.
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20 Naturally-occurring Amino Acids
Acidic D, E, (C, Y) Basic K, R,
H Hydrophobic I, L, V, W, F Polar S, T, N,
Q Other G, A, M, P
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Overview of Protein Purification Types
of Separations Protein Properties Protein
Inactivation/Stabilization
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Protein Analysis and Purification Analytical
Separations Gel-electrophoresis IEF 2D-gels Prepa
rative Separations Various chromatographic
methods
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Total E. coli Proteins - 2-Dimensional Gel
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Main Types of Molecular Interactions
Hydrogen Bonds N H - - - - N N-H
N low temperature high
temperature N H - - - - O C
strength is very dependent on geometry
donor acceptor
and distance (2.6-3.1 A) Hydrophobic
Interactions (waxy residues Ileu, Leu, Val, Phe,
Trp) high salt high
temperature low salt Ionic
Interactions (charged residuesAsp- Glu- S-
Lys Arg His)
low ionic
strength high ionic strength
H
H
H
H
H2O
H
H
H
H
Cl-
Na
...
-

-

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Variables that Affect Molecular
Forces Temperature Ionic strength Ion
type Polarity of solvent (dielectric constant) pH
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Protein Properties - Handles for Fractionation

• Size (110 Da/amino acid residue)
• smallest most proteins largest
• Amino acids 30 100 1,000
  15,000
• MW (kDa) 3.3 11 110
  1,600
• Multi-subunit complexes can contain 5-30
  subunits
• Shape
• globular (sphere) asymmetric (cigar)
• Effects frictional properties, effective radius,
  movement through pores

Centrifuge
Gel filtration
Elutes earlier Appears larger
Sediments slower Appears smaller
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Protein Properties - Handles for Fractionation

• Net charge
• Ionizable group pKa pH2 pH7
  pH12
• C-terminal (COOH) 4.0 oooooooo-------------------
  ---------------------
• Aspartate (COOH) 4.5 oooooooooo------------------
  -------------------
• Glutamate (COOH) 4.6 ooooooooooo-----------------
  -------------------
• Histidine (imidazole) 6.2 oooooooooo
  oooooooooo
• N-terminal (amino) 7.3 ooooooooooo
  ooooooo
• Cysteine (SH) 9.3 ooooooooooooooooooooooo--------
  ---------
• Tyrosine (phenol) 10.1 oooooooooooooooooooooooooo
  -------------
• Lysine (amino) 10.4 ooooo
  ooo
• Arginine (guanido) 12.0
  o
• Isoelectric point
• pI pH where protein has zero net charge
• Typical range of pI 4-9
• Charge distribution

versus
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Protein Properties-Handles for Fractionation

• Hydrophobicity
• Hydrophobic residues usually are buried
  internally
• The number and distribution on the surface
  vary
• Can use Hydrophobic Interaction Chromatography
• Solubility
• Varies from barely soluble (ltmg/ml) to very
  soluble (gt300 mg/ml)
• Varies with pH, ionic strength/type, polarity
  of solvent, temperature
• Least soluble at isoelectric point where there
  is least charge repulsion
• Ligand and metal binding
• Affinity for cofactors, substrates, effector
  molecules, metals, DNA
• When ligand is immobilized on a bead, you have
  an affinity bead

hydrophobic patch
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Separation Processes that can be Used to
Fractionate Proteins
Separation Process
Basis of Separation Precipitation ammonium
sulfate solubility polyethyleneimine
(PEI) charge, size isoelectric
solubility, pI Chromatography gel filtration
(SEC) size, shape ion exchange (IEX)
charge, charge distribution
hydrophobic interaction(HIC) hydrophobicity DN
A affinity DNA binding site immunoaffinity
(IAC) specific epitope chromatofocusing
pI Electrophoresis gel electrophoresis (PAGE)
charge, size, shape isoelectric focusing
(IEF) pI Centrifugation sucrose gradient
size shape, density Ultrafiltration ultrafiltratio
n (UF) size, shape
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Typical Protein Purification Scheme
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Protein Inactivation/StabilizationBuffers
Solution Components
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Protein Sources for Purification Traditional
natural sources Bacteria, animal and plant
tissue Cloning recombinant proteins into
overexpression vector/host systems for
intracellular production (E. coli the most
used) In vitro protein synthesis
Transcription/translation systems
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Total E. coli Proteins - 2-Dimensional Gel
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Determining the protein sequence from
gel (proteomics)
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What You Can Learn from Amino Acid Sequence 1.
Molecular weight of the polypeptide chain 2.
Charge versus pH Isoelectric point 3.
Extinction coefficient 4. Hydrophobicity
membrane spanning regions 5. Potential
modification sites 6. Conserved motifs that
suggest cofactor affinityWhat You Cant Learn
from Amino Acid Sequence 1. Function 2.
3-Dimensional structure Shape 3.
Multi-subunit features 4. Ammonium sulfate
precipitation properties 5. Surface features
(hydrophobic patches, charge
distribution, antigenic sites)Conclusion
Protein Purification is still very empirical!
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Engineering Proteins for Ease of Purification and
Detection Once you have a gene cloned and can
over-express the protein, you can alter protein
to improve the ease of purification or
detection You can fuse a tag to the N-or C-
terminus of your protein You can decide to
remove the tag or not Basic strategies Add
signal sequence that causes secretion into
culture medium Add protein that helps the
protein refold and stay soluble Add sequence
that aids in precipitation Add an affinity
handle (by far the most used is the His-tag) Add
sequence that aids in detection
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CSH Protein Course -Sigma32 Purification
MW A B C D E F
G A/3 B/3 D/3
225 50 35 10 kDa
bb s32