Download Protein Purification

Introduction
Techniques of Protein and
Nucleic Acid Purification
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Biochemical investigations usually require pure components
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typical cell contains thousands of different substances
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many biomolecules have similar physical & chemical properties
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biomolecules may be unstable and/or present in vanishingly small
quantities
Voet Biochemistry: Chapter 6, Pages 127 - 151
Voet Fundamentals: Chapter 5, Pages 94 - 103
⇒ Purification of biomolecules is a formidable task
⇒ would be considered unreasonably difficult by most synthetic
chemists
Lecture 3
Biochemistry 2000
Slide 1
General Protein Purification
Strategy
Lecture 3
Procedure
Solubility
1. Salting in
2. Salting out
Ionic Charge
1. Ion exchange chromatography
2. Electrophoresis
3. Isoelectric Focusing
Polarity
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1. Adsorption chromatography
2. Paper chromatography
3. Reverse-phase chromatography
4. Hydrophobic interaction chromatography
1. Dialysis and ultrafiltration
2. Gel electrophoresis
3. Gel filtration/Size exclusion chromatography
Binding specificity
1. Affinity chromatography
Biochemistry 2000
Adjust solution to just below the point the solubility of
the protein of interest:
− change ionic strength (add salt)
− Change polarity (add organic solvent)
− Change pH
− Change temperature
Precipitate proteins other than the protein of interest
Separate soluble and insoluble material by centrifugation
or filtration
Typically the first step in a protein purification
(a) mixture of 3 protein - white, grey,
black
Molecular size
Lecture 3
Slide 2
Solubility-based Purification
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Characteristic
Biochemistry 2000
Slide 3
(b) solution altered – black protein
precipitates (supernatant removed)‫‏‬
(c) solution altered again – grey protein
precipitate (white protein remains in
supernatant)‫‏‬
Lecture 3
Biochemistry 2000
Slide 4
Effects of Ionic Strength
Salting in:
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protein solubility increases with ionic
strength (I) (at low ionic strength)
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Due to shielding of protein charges
Chromatography
Salting in
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Salting out
Salting out:
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protein solubility decreases with
increasing ionic strength (at high I)
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Due to competition for molecules of
solvation
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Biochemistry 2000
Slide 5
Lecture 3
Ion Exchange
Chromatography (IEC)‫‏‬
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Substances interacting with stationary phase are retarded
Continuous process in which sample is subject to repeated,
identical separations
classified according to retarding force (eg. ion exchange,
affinity, size exclusion)‫‏‬
Biochemistry 2000
Slide 6
IEC and Stepwise Elution
Stationary phase: beads uniformly coated with charged groups
Mobile Phase: ions and proteins binding reversibly to stationary phase
through electrostatic interactions
R+-Ion- + Protein-
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percolated through a column containing a “stationary” phase
(solid)
Most powerful separation technique in Biochemistry
Salting out is the basis of one of the most
common purification protocols
Lecture 3
Mixture of substances is dissolved in “mobile” phase (liquid)
R+-Protein-
+ Ion-
Strength of binding depends upon pH and the identity and concentration
of ions in solution
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First, chose conditions where protein binds to stationary phase
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Then, elute protein by changing to conditions where protein dissociates
Anion exchange – anions in mobile phase bind cationic stationary phase
Cation exchange – cations in mobile phase bind anionic stationary phase
Typically the first chromatography step in a purification
Lecture 3
Biochemistry 2000
Slide 7
Lecture 3
Biochemistry 2000
Slide 8
Size Exclusion
Chromatography (SEC)‫‏‬
SEC
Also: gel filtration chromatography or molecular sieve
chromatography
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Separation based upon molecular size (and shape)‫‏‬
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Stationary phase contains pores that span a narrow
size range
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Large molecules cannot enter small pores and flow
rapidly through column
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Smaller molecules enter some or all pores (depending
upon their size) and traverse the column more slowly
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Quantitative retardation of smaller molecules
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Typically last chromatography step in a purification
Lecture 3
Biochemistry 2000
Slide 9
Lecture 3
SEC and Molecular Weight
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Linear relation between log MW and ratio of elution and void volume
(Ve/Vo) (except for highly asymmetric proteins)
Elution volume Ve:
Volume to elute
protein after it first
contacted column
Void volume Vo:
volume of solvent
within column, i.e.
total column volume
minus volume of
stationary phase
Biochemistry 2000
Slide 10
Affinity Chromatography
Quantitative determination of molecular mass (MW) by SEC
Lecture 3
Biochemistry 2000
Slide 11
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many proteins tightly bind specific
molecules (ligands) non-covalently
Attaching (covalently) the ligand to a matrix
allows protein purification by specific
binding
Only the desired protein(s) in an impure
mixture will bind to the matrix
Protein can be eluted in pure form by
altering conditions, e.g. by adding large
amount of free ligand
Single-step purification in favorable cases
Lecture 3
Biochemistry 2000
Slide 12
Electrophoresis
FPLC / HPLC
Fast Protein Liquid Chromatography (FPLC)
migration of ions in an electric field
High Performance Liquid Chromatography (HPLC)
Fast, easy and cheap separation method
• Improved separation (higher resolution &
sensitivity)
Not generally usable for large scale separation
and recovery of samples
• High-resolution columns with more and smaller,
incompressible beads
Migration of ions including proteins depends upon both
molecule charge (q) and the frictional coefficient (f):
•
• Automation using pumps reaching a pressure
of up to 5000 psi
Felectric = qE
(E – electric field)
(v – velocity)
Ffriction = vf
Felectric = Ffriction = qE = vf
• faster purification
=> electrophoretic mobility: μ = v/E = q/f
Frictional coefficient depends on size, shape and solution viscosity
•
Lecture 3
Biochemistry 2000
Slide 13
Lecture 3
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Slide 14
Discontinuous Gel
Electrophoresis
Gel Electrophoresis
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Biochemistry 2000
Gel is porous and typically made of polyacrylamide (< 200 kD) or
agarose (<10000 kD)
PAGE‫ = ‏‬Polyacrylamide Gel Electrophoresis
Separation is based upon both:
Electrophoretic mobility & Gel Filtration
large proteins are retarded relative to
small (opposite of SEC)‫‏‬
Upper “stacking” gel:
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large pores & Glycine Buffer with lower pH (6.9)
Lower “resolving” gel:
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Small pores & Glycine Buffer with higher pH (8.8)
⇒ Stacking gel generates narrow sharp “bands”
Why?
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Pore
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Lecture 3
Biochemistry 2000
Slide 15
Glycines are neutralized in stacking gel, i.e. have
only small net charge
Local electric field increases and concentrates
sample
Once in running gel the electric field is constant
Lecture 3
Biochemistry 2000
Slide 16
Discontinuous Gel
Electrophoresis
Negative
Stacking
Gel pH 6.9
SDS-PAGE
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Glycine
Proteins
& Cl-
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Narrow
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Separated
proteins
Resolving
Gel pH 8.8
Most proteins bind SDS at a constant ratio (~ 1
SDS molecule per 2 residues)‫‏‬
Swamps native charge of protein
Results in average constant charge density
AND similar shape for all proteins
⇒ Separates based upon size only
Positive
+
Glycine: H3N+CH2COOLecture 3
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SDS is a detergent that denatures proteins and
binds strongly to proteins
+
H2NCH2COO- + H+
Biochemistry 2000
Slide 17
Lecture 3
1. Coomassie Stain:
• Denatures protein and binds to hydrophobic core
• Excess can be washed away
• Detection limit is 0.1 μg
Application:
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Size estimation: mobility depends linearly
on log MW, standard curve required
2. Silver Stain:
• up to 50x more sensitive
• more expensive and difficult to apply
Detection of non-covalenty associated
subunits resulting in multiple bands since
subunits dissociate when protein is
denatured
Typically reduction of disulfide bridges
with β-mercaptoethanol (reducing agent)
Lecture 3
Biochemistry 2000
Slide 18
Protein Detection Methods
SDS-PAGE
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Biochemistry 2000
Coomassie stained
SDS-PAGE of
Affinity Chromatography
protein purification
Slide 19
Lecture 3
Biochemistry 2000
Slide 20
Assays
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All purification protocols require a means
to quantitatively detect the
macromolecule
Assay must be specific as many
macromolecules have closely similar
properties
Functional assays are most common
Enzymatic activity, specific binding,
observed biological activity,
immunochemistry
Steps 1 & 2 – Specific binding of
protein of interest to known antibody
(assay)‫‏‬
Steps 3 & 4 – Detection of binding
using second known antibody
Lecture 3
Biochemistry 2000
Slide 21