Introduction to Protein Purification Principles of Chromatography Affinity Chromatography Richard Bu

1
Introduction to Protein PurificationPrinciples
of ChromatographyAffinity Chromatography
Richard BurgessUniversity of Wisconsin-Madisonb
urgess_at_oncology.wisc.edu June 12, 2009Colorado
State University
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Typical Protein Purification Scheme
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Purification Summary Table

. Fraction
Total Total Rel. Specific
Yield Protein (mg)
Activity () Activity () . Extract
12,000 100
1 100 75 AS pptn (45-50) 1,800
75 5
75 80 IEC (pooled peak) 240
60 30 60
75 Gel filtration (peak) 36 45
150 45

fold-purif final yield Pure standard
150
. From 100 g wet weight
cells Key terms overall yield, step yield,
purity, fold-purification, specific activity,
relative specific activity,
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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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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 (Pressure)
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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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Titration Curve (Charge vs pH) of Sigma32
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Protein Properties-Handles for Fractionation

• Hydrophobicity
• Hydrophobic residues usually are buried
  internally
• The number and distribution on the surface
  vary
• 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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Protein Properties-Handles for Fractionation

• Reversible association
• for example, RNA polymerase
• Post-translational modifications
• Carbohydrates, lipids, phosphates, sulfates
• Can be very useful purification handles
• E.g. Use of plant lectins to bind certain
  glycoproteins
• Specific sequence or structure
• Precise geometric presentation of amino acids
  on surface of a protein
• Epitope for binding to a specific antibody
  use immunoaffinity column
• Binding site for another protein use protein
  affinity column

Monomer (0.3M NaCl) Dimer (0.05 M NaCl)
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Post-Translational Modifications

• Addition of non-protein material
• Carbohydrates are added, leading to 0- and
  N-glycosylation
• Phosphates can be added by kinases /removed by
  phosphatases
• AMP, ADP-ribose, or sulfate can be added
• Lipids can be added
• at myristoylation sites
• isoprenylation of C-terminal cysteines
• Acetylation or methylation of lysines in
  histones, etc
• Acetyl transferases/deacetylases
• Methylases/demethylases
• Several relatively rare amino acid modifications
  (hydroxyproline)

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Separation Processes that can be Used to
Fractionate Proteins
Separation Process
Basis of Separation Precipitation ammonium
sulfate solubility polyethyleneimine
charge, size isoelectric solubility,
pI Chromatography gel filtration (SEC) size,
shape ion exchange (IEX) charge, charge
distribution hydrophobic
interaction(HIC) hydrophobicity DNA 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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General Protein Purification Strategy
Must have a convenient assay to follow
purification Choose a starting material rich in
your protein Take precautions to minimize damage,
inactivation or loss Use the minimal number of
steps Remove bulk of material quickly Avoid
duplication, dialysis and delay Generally use in
order precipitation - ion exchange - affinity -
sizing Use high-resolution steps where
possible Pool narrowly for purity, broadly for
yield Can use a column to purify by subtraction
(affinity depletion) Optimize procedure before
you submit paper to my journal (PEP)
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Ammonium Sulfate Precipitation Curve
LogS b-Ks(AS)
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Polyethyleneimine (PEI) Precipitation
Differences from Ammonium Sulfate
Precipitation 1. Titration, not salting
out 2. Takes less PEI if dilute the extract 3.
Only precipitates acidic molecules 4. Can elute
from precipitate (it is almost like soluble
DEAE 5. Must be removed or it will
re-precipitate at low salt
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Strategies for the Use of PEI Strategy A
Precipitate with PEI at high salt (1.0 M
NaCl) Remove nucleic acids Leave
almost all proteins in the supernatant Strategy B
(neutral or basic proteins) Precipitate with
PEI at low salt (0.1 M NaCl) Remove nucleic
acids and acidic proteins Leave enzyme of
interest in the supernatant Strategy C (acidic
proteins, such as E. coli RNA polymerase) Precipit
ate with PEI at low salt (0.1 M NaCl)
Precipitate nucleic acids and acidic proteins
Wash some proteins out of pellet with
medium salt (0.4 M NaCl) Elute desired enzyme out
of the pellet with higher salt (0.9 M NaCl) Leave
nucleic acids in the pellet Follow with a 60
saturated AS precipitation to remove PEI
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Column Chromatography Types Adsorption Ion
exchange Anion Exchange - Q, DEAE Cation
Exchange - S, SP, Phosphocellulose,
BioRex70 Mixed Affinity General -
hydroxyapatite, triazine dyes, IMAC -
immobilized metal affinity chromatography Specifi
c - bound ligand (cofactor, substrate) Hydrophobi
c - phenyl, octyl Immuno - monoclonal
antibody Protein - another protein DNA - DNA,
specific and non-specific Gel Filtration - size
exclusion Adsorption/desorption versus
differential movement down the column Low
pressure chromatography versus HPLC
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Ion Exchange Chromatography - General
Principles Negatively charged proteins will bind
to positively charged column materials (anion
exchangers), while positively charged or neutral
proteins will flow through the column. The more
negatively charged, the tighter it will bind and
the higher the salt it will take to elute it from
the column Some highly charged proteins, where
the charge is not evenly distributed, can bind to
both anion and cation exchangers (e.g.,
sigma32). Some relatively uncharged proteins
wouldnt bind to either type column. Use two
different columns in tandem, collect flow through

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General Considerations Batch versus
column mode Washing, de-fining, pouring,
equilibrating Capacity - milli-equivalents(meq)/ml
(determine by titrating with acid or
base mg BSA bound/ml capacity
can depend on MW of protein Loading washing,
eluting, stripping Elution
Conditions Isocratic elution step elution,
gradient elution Effect of varying flow rate,
gradient steepness Protein elution usually
cooperative, elutes sharply
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Could also vary salt at a given pH to test what
salt to use or vary amount of supernatant
added to test capacity of resin
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Other Considerations With new
smaller beads, can run columns much faster (in
minutes) and still have high resolution. This and
the advent of automated loading and elution
gradient program control, means that one can
load, and run multiple identical columns, thus
using a smaller column and saving money on
expensive column resins Remember, anions bind to
DEAE (e.g., PO4- eluting) EDTA can bind to, elute
from MonoQ at about 0.25 M NaCl Purification by
subtraction when a 0 (e.g., IgG at pH 6.9 on
DEAE-cellulose IgG is in flow through while most
of the rest of the proteins in the serum bind to
the column). Trailing of peaks with isocratic
elution is due to a distribution of protein
dissociation constants. Can sharpen peak by
gradient elution Interference from nucleic acids
- decreases capacity
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Some Relative Costs Whatman - DE
52, traditional resin (0.40/ml),
DEAE-cellulose capacity 130 mg BSA/ml GE
Healthcare (Pharmacia) Q-Sepharose FF (0.65/ml)
CL agarose, 6, 50-150 mm, capacity
120 mg BSA/ml PerSeptive BioSystems (Applera) -
POROS 50 HQ bulk (3/ml), 50 mm GE Healthcare
(Pharmacia) - Mono Q (1-ml column - 855)
polyether, 10 mm, 65 mg BSA/ml - but can
reuse many times!
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(Amersham)
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Types of Affinity Chromatography Ligand
(ATP, NAD, estrogen, etc) Takes advantage
of specific binding properties Uses small
molecule substrates, cofactors or mimics General
Takes advantage of general
affinities HIC (hydrophobic interaction
chromatography) IMAC (immobilized metal affinity
chromatography) Protein-Protein DNA
(non-specific and specific) Antibody
(immunoaffinity)
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Hydrophobic Interaction Chromatography
(HIC) Mildly hydrophobic groups attached to
column (butyl, octyl, phenyl) Takes advantage
of the hydrophobic patches on surface of soluble
proteins Load under high salt condition (e.g., 1
M AS) to salt out protein onto column (the high
salt strengthens the hydrophobic interaction) Can
go directly from ammonium sulfate precipitate to
HIC column Elute with a decreasing salt gradient.
Least hydrophobic proteins elute early, followed
by more hydrophobic proteins.
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• Protein-DNA Binding Considerations
• A. Specific binding proteins interact with DNA
  both specifically and non-specifically
• For example, E. coli RNA polymerase
• Specific contacts H-bonds between protein
  residues and basepairs
• Non-specific contacts Ionic interactions of
  positive residues with phosphate groups
• Specific KA 1010 M-1 Non-specific KA 106
  M-1
• Specificity ratio KAsp/KAnonsp 1010/106 104
• Occupancy (number of sites)(K)
• On a DNA the size of pBR322 (5000bp) 10,000
  non-specific sites
• Specific (1 site)(1010) 1010 Non-specific
  (104 sites)(106) 1010
• Therefore there are equal amounts of protein
  occupying specific and non-specific sites!!

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DNA-Affinity Chromatography
(Specific) Especially important for purifying
non-abundant specific DNA binding proteins for
transcription, replication, recombination
(often less that 0.01 of total
protein) Nature of the Problem Multiple
factors, rare proteins Often dont have
good individual assays But do have gel
shift and footprinting assays Kadonaga Method How
to get specific binding, prevent unwanted
non-specific binding? Increase concentration of
specific sites Use competitor DNA
lacking specific sites. e.g., poly(dAT) Increase
salt to weaken non-specific binding Elution
Most often elute with increasing salt

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Preparation of a DNA Affinity Column
(Kadonaga and Tjian, PNAS 83 5889, 1986)

• Synthesize complementary oligos (14-20 b)
  containing desired specific sequence and 4 base
  overhang
• Anneal oligos
• Add 5 PO4 with ATP and T4 Polynucleotide Kinase
• Ligate with T4 DNA ligase to produce concatamers
  (30-mers)
• Couple to CNBr-activated Sepharose (150mg DNA/ml
  resin)
• Apply extract containing competitor (5-50mg
  dIdC/ml), wash 50 mM NaCl, and elute with 0.5 M
  NaCl

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Gentle Immunoaffinity Chromatography Using
Polyol-Responsive Monoclonal Antibodies for
Purification of Biologically Active, Labile,
Multi-subunit Protein ComplexesNancy Thompson
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Immunoaffinity Chromatography Pros The most
powerful fractionation step known, capable of
10,000-fold purification in one step Cons Often
difficult to elute bound protein, requires
extreme pHs or use of chaotropic reagents
Solution Find monoclonal antibody that binds
tightly but releases under gentle conditions
Polyol-Responsive Monoclonal Antibodies !!!!
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Practical Tips on Immunoaffinity
Chromatography Generally expensive unless you
make your own MAbs Wash well before use to remove
MAb that has leached off during storage Avoid
DTT (but can use 0.1 mM briefly) to prevent
reduction of essential disulfide bonds and loss
of MAb from column Can bind on-column but dont
load too fast (lt 1 ml/min) Elute bound protein
slowly (not usually a problem when using
40 polyol) Must reuse column many times to
purify large amounts of protein After use can
clean up column by stripping off residual bound
protein briefly with 2 ml of 2 M KSCN (can
inactivate MAb) Storage of column (add 0.01
NaAzide, store at 4oC for months) Probably could
store in 50 glycerol at -20oC, can even survive
freezing at -70oC
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Binding and Capacity Considerations Dissociatio
n constant of mAb and antigen from 10-6 to 10-12
M Find that immobilizing about 2.5 mg mAb/ml of
resin is optimal Ligand Efficiency (LE)
Defined as the amount of antigen bound compared
to the theoretical amount that could be bound
assuming each mAb binds two antigens and 100 of
mAb is active For example, if IgG 150 kDa
and Enzyme 100 kDa, 2.5 mg of mAb bound to a
1-ml column can bind (2 x 100/150) x
2.5 3.3 mg enzyme if LE is 100 Often LE is
only 10-30 Orientation of the mAb is important
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• Why use Monoclonal Antibodies for
  Immunoaffinity Chromatography?
• Takes only 50-100 ug of antigen, need not be pure
• Indefinite supply of reproducible antibody
• All IgG is specific whereas most of IgG in a
  polyclonal Ab mixture is non-specific
• mAb has homogeneous binding properties, while
  polyclonal Ab is mixture of Abs with different
  binding constants

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General Procedure for the Isolation and Use of
Polyol-Responsive Monoclonal Antibodies Inject
mouse with immunogen Perform fusion Screen
hybridomas for positives Screen positives by
ELISA-elution with salt/polyol Clone
hybridoma Optimize elution conditions by
ELISA-elution Prepare antibody Prepare
immunoaffinity column Purify antigen
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Treat with Salt/Polyol
React Enzyme-Conjugated Secondary Antibody
Ag
Ag
Ag
Ag
Ag
Ag
Substrate
Color
React with Substrate
Ag
Ag
Ag
Ag
Ag
Ag
An MAb is considered to be polyol-responsive if
the absorbance reading of the wells treated with
polyol/salt are 50 or less than the wells
treated with buffer alone.
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ELISA-Elution Screening of Hybridomas from Master
Wells
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ELISA-Elution with MAb NT73 -
Combinations of NaCl and Various Polyols
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ELISA-Elution with MAb NT73
Salt Concentration (M)
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NT73 Immunoaffinity on Crude Extract
b? b
s70
a
w
MW ON FT 0.5 M NaCl
Wash 0.7 M NaCl40 Propylene Glycol
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Ann and Dick Burgess, on mini-sabbatical in
Melbourne, Australia, 2004
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Ann Dick Burgess, 2004
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Generalities of Polyol-Responsive MAbs
- Can routinely isolate suitable MAbs (About 5
of positive hybridomas) - Can easily optimize
eluting conditions - MAbs are high affinity -
Can elute under mild conditions - High recovery
of active protein - Not much more complicated
than normal MAb isolation - Used successfully on
many different proteins
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Use gentle immunoaffinity chromatography to
purify endogenous and associated proteins from
cell extracts
cell extract
Control IgG column
Anti-ERRa PR-MAb column
SEC or IEC
Collect fractions
Elute with SaltPolyol
Collect flow through
Run SDS-PAGE Stain gel with Coomassie
ERRa
ERRa
WB with anti-ERRa MAb
(Jenny Lamberski)
Prep for MS
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Yeast RNA Polymerase II
Structure (Cramer, Thompson, Burgess,
Kornberg, et al, Science 288 640, 2000)
Won Nobel Prize for this work 2006
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References to Polyol-Responsive Monoclonal
Antibodies Burgess RR and Thompson NE. Advances
in gentle immunoaffinity chromatography. Current
Opinions in Biotech 2002, 13 304-308 Bergendahl,
V., Thompson NE, Foley, K., Olson, B., and
Burgess, R.R. A cross-reactive polyol-responsive
monoclonal antibody useful for isolation of core
RNA polymerase from many bacterial species.
Protein Expression Purification 2003, 31
155-160 Bergendahl, V. Glaser, B., and Burgess,
R.R. A fast western blot procedure improved for
quantitative analysis by direct fluorescence
labeling of primary antibodies. J. Immunol.
Methods 2003, 277 117-125 Thompson, NE, Arthur,
TM, and Burgess, RR. Development of a new epitope
tag for the gentle purification of proteins by
immunoaffinity chromatography application to
epitope-tagged green fluorescent protein. Analyt
Biochem 2003, 323 171-179 Duellman, SJ,
Thompson, NE, and Burgess, RR. An epitope tag
derived from human transcription factor IIB
(TFIIB) that reacts with a polyol-responsive
monoclonal antibody. Protein Expression
Purification 2004, 35 147-155 Thompson, NE,
Foley, KM, Burgess, RR. Antigen-binding
properties of monoclonal antibodies reactive with
human TATA-binding protein and use in
immunoaffinity chromatography. Protein Expression
Purific 2004, 36 186-197 Probasco, MD,
Thompson, NE, and Burgess, RR, Immunoaffinity
purification and characterization of RNA
polymerase from Shewanella oneidensis, Protein
Expression and Purification, 55 23-30, 2007.
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Cross-reactivity of Selected RNAP-Specific MAbs
Bacterium 2G10 3RD3 NT73
6RN3 4RA2 1RS1 8RB13
(s70) (s70)
(b) (s54) (a) (sS)
(b) Escherichia coli

Klebsiella pneumoniae
-
Salmonella typhimurium

Shigella boydii

Serratia marcescens

Vibrio parahemolyticus
-
Pseudomonas aeruginosa
-
Rhodobacter spheroides
- -

- Agrobacterium tumefaciens -
- -
- Bordetella pertussis
-
Staphylococcus aureus
-
Streptococcus faecalis
- -
- - Bacillus subtilis
- -
Borrelia burgdorferi
- -
- Shewanella oneidensis
-
- Anabena sp.
-
-
Nancy Thompson, Kit Foley Red
Polyol-responsive MAb
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A New Epitope Tag Softag1 Identification of
Epitope for the Polyol-Responsive Mab NT73 to the
C-Terminus of the b Subunit of RNA Polymerase
(Thompson, Arthur, Burgess, Anal. Biochem,
323171-179, 2003)
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NT73 Immunoaffinity Purification of
Epitope-Tagged GFP (SDS gel analysis)
On FT Eluate
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Properties of Softag 1 as an Epitope Tag 1. High
affinity 2. Allows gentle immunoaffinity
purification of any fused protein with full
retention of biological activity 3. Allows
isolation of any proteins associated with target
protein so can help identify protein binding
partners 4. Produces homogeneous protein suitable
for crystallization 5. Can be attached to either
C-terminus or N-terminus 6. Small (13 amino acids
or less) 7. Not highly charged or hydrophobic 8.
Demonstrated to work on several proteins (GFP and
TFIIB) 9. MAb NT73 is commercially available
(NeoClone)
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ST3K2Q-tagged GFP on IIB8 Column

• Induced GFP-ST3K2Q protein expression
• Ran crude extract over IIB8 immunoaffinity
  column column fluoresced!
• Washed column
• Eluted fusion protein with TE0.75M AS 40
  propylene glycol
• Fractions fluoresced!
• (Sarah Duellman)

Fractions 3-9
S FT TEN TE.5 M
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Chromatography Components Column Length
and width Material (glass, plastic, stainless
steel) Plumbing (has improved dramatically in
last 20 yrs) E.g., flow adaptors, low dead
volume New configurations - radial flow,
membranes
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Chromatography Components
Resin Material (cellulose, agarose,
polyacrylamide, silica (SiO2), synthetic
polymers) Shape Pore size Particle size
Particle uniformity Capacity (equivalents,
accessibility) Equilibrium (affected by the bead
size) affects maximum flow rate Packing
(pack under flow rate at which you plan to run
column) Washing, recycling
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Chromatography Components
Sample Volume (small for gel filtration, can
be very large for ion exchange) Protein amount,
purity, viscosity, clarity Elution Choices
Isocratic versus step versus gradient Gravity
feed or pump Buffer composition (pH, ionic
strength, ion type Can vary more than one at
a time) Flow rate, (cm/hr x cross section
cm3/hr Steepness, volume Temperature
(if use jacketed column) Direction (down or up)
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Chromatography Problems Poor
packing Poor loading Dead volume Running dry
(Importance of understanding
hydrostatics) If move from cold room to room
temperature - bubbles form as solubility of
gas is decreased Cost of new materials (up to
1000/1 ml column!) Non-specific adsorption to
resin - protein loss Inadequate recycling or
cleaning- protein contamination from
previous run carryover
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Chromatography Equipment Recorder 1
or 2 channel, multi-channel Data storage and
analyzer Store, rescale, subtract blank,
overlay, ratios Fraction collector
Computer control Newer systems use computer to
program run, monitor status, store methods,
collect data, analyze data (e.g., relative area
under peaks), send data to printer. Allows
highly reproducible runs, automatic multiple
applications
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Ni2-NTA Purification of His6-tagged
Proteins Bind at pH 7.9, 5 mM imidazole to
prevent weak binding of His or Cys-containing
proteins 300 mM NaCl to prevent ionic binding
can bind in presence of 6 M GuHCl or 8 M urea so
can purify protein denatured and then refold it
(you can also refold it on the column) Wash
20-80 mM imidazole. Can wash with 60
isopropanol to remove endotoxins, residual
detergents, some non-specifically bound
proteins Elute 3 different methods 300-800
mM imidazole (most common way) EDTA to remove
Ni2 from NTA lower pH (less that pH 5.9)
where the imidazole group of Histidine becomes
protonated
63
Interaction Between Neighboring His residues on
the His6-tag and Ni-NTA Matrix
64
Structure of Imidazole and Histidine
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Ni2-NTA Purification of His6-tagged
Proteins Additional Tidbits Ni-NTA binding of
His6-tagged proteins (E. Hochuli in
1987) Aggregate (e.g., dimer) requires higher
imidazole to elute 10-10 M Ni leaches off
column, can accelerate air oxidation of
DTT Unlike what Qiagen says, you can use
(NH4)2SO4 with Ni columns Sometimes you find
bound DnaK and can wash it off with ATPMg2 One
protein in E. coli binds very tightly (SlyD, 27
kDa, very His-rich) (Roof et al., Molec.
Microbiol. 25 1031, 1997) Can use Cobalt (Talon
columns) or Zinc in place of Nickel Often (but
not always) proteins can be used without removing
His-tag Thiol reducing agents form metal
sulfides, cause column to turn brown Can avoid
by using TCEP
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Courtesy of Paul Blommel (B. Fox Lab)
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My Background1. PhD in Biochemistry and
Molecular Biology, 1969 2. James D. Watson
Professor of Oncology in McArdle Laboratory for
Cancer Research 3. Research focused on RNA
polymerase and transcription factors, their
purification, characterization and role in
regulation of gene expression4. Founded
directed UW Biotechnology Center (1984-96)5.
Instructor, Onc. 675 Protein Purification
(1986-present)6. Editor-In-Chief of journal,
Protein Expression and Purification
(1994-present)7. Instructor and Chair, Cold
Spring Harbor Course on Protein Purification
and Characterization (1992-present)8. Editor of
Guide to Protein Purification Methods in
Enzymology, in prep 2009 (with Dr. M.
Deutscher)9. I love to purify proteins!