Download Techniques of Protein and Nucleic Acid Purification

Techniques of Protein and
Nucleic Acid Purification
Voet Biochemistry: Chapter 6, Pages 127 - 151
Voet Fundamentals: Chapter 5, Pages 94 - 103
Lecture 3
Biochemistry 2000
Slide 1
Introduction
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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
⇒ Purification of biomolecules is a formidable task
⇒ would be considered unreasonably difficult by most synthetic
chemists
Lecture 3
Biochemistry 2000
Slide 2
General Protein Purification
Strategy
Characteristic
Procedure
Solubility
1. Salting in
2. Salting out
Ionic Charge
1. Ion exchange chromatography
2. Electrophoresis
3. Isoelectric Focusing
Polarity
1. Adsorption chromatography
2. Paper chromatography
3. Reverse-phase chromatography
4. Hydrophobic interaction chromatography
Molecular size
1. Dialysis and ultrafiltration
2. Gel electrophoresis
3. Gel filtration/Size exclusion chromatography
Binding specificity
1. Affinity chromatography
Lecture 3
Biochemistry 2000
Slide 3
Solubility-based Purification
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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
(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
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
Salting in
Salting out
Salting out is the basis of one of the most
common purification protocols
Lecture 3
Biochemistry 2000
Slide 5
Chromatography
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Mixture of substances is dissolved in “mobile” phase (liquid)
percolated through a column containing a “stationary” phase
(solid)
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)‫‏‬
Most powerful separation technique in Biochemistry
Lecture 3
Biochemistry 2000
Slide 6
Ion Exchange
Chromatography (IEC)‫‏‬
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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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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
IEC and Stepwise Elution
Lecture 3
Biochemistry 2000
Slide 8
Size Exclusion
Chromatography (SEC)‫‏‬
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Also: gel filtration chromatography or molecular sieve
chromatography
Separation based upon molecular size (and shape)‫‏‬
Stationary phase contains pores that span a narrow
size range
Large molecules cannot enter small pores and flow
rapidly through column
Smaller molecules enter some or all pores (depending
upon their size) and traverse the column more slowly
Quantitative retardation of smaller molecules
Typically last chromatography step in a purification
Lecture 3
Biochemistry 2000
Slide 9
SEC
Lecture 3
Biochemistry 2000
Slide 10
SEC and Molecular Weight
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Quantitative determination of molecular mass (MW) by SEC
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
Lecture 3
Biochemistry 2000
Slide 11
Affinity Chromatography
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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
FPLC / HPLC
Fast Protein Liquid Chromatography (FPLC)
High Performance Liquid Chromatography (HPLC)
• Improved separation (higher resolution &
sensitivity)
• High-resolution columns with more and smaller,
incompressible beads
• Automation using pumps reaching a pressure
of up to 5000 psi
• faster purification
Lecture 3
Biochemistry 2000
Slide 13
Electrophoresis
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migration of ions in an electric field
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Fast, easy and cheap separation method
Not generally usable for large scale separation
and recovery of samples
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Migration of ions including proteins depends upon both
molecule charge (q) and the frictional coefficient (f):
(E – electric field)
Felectric = qE
(v – velocity)
Ffriction = vf
Felectric = Ffriction = qE = vf
=> electrophoretic mobility: μ = v/E = q/f
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Frictional coefficient depends on size, shape and solution viscosity
Lecture 3
Biochemistry 2000
Slide 14
Gel Electrophoresis
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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)‫‏‬
Pore
Lecture 3
Biochemistry 2000
Slide 15
Discontinuous Gel
Electrophoresis
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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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
-
Glycine
Proteins
& Cl-
Narrow
Separated
proteins
Resolving
Gel pH 8.8
Positive
+
Glycine: H3N+CH2COOLecture 3
Biochemistry 2000
+
H2NCH2COO- + H+
Slide 17
SDS-PAGE
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SDS is a detergent that denatures proteins and
binds strongly to proteins
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
Lecture 3
Biochemistry 2000
Slide 18
SDS-PAGE
Application:
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Size estimation: mobility depends linearly
on log MW, standard curve required
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 19
Protein Detection Methods
1. Coomassie Stain:
• Denatures protein and binds to hydrophobic core
• Excess can be washed away
• Detection limit is 0.1 μg
2. Silver Stain:
• up to 50x more sensitive
• more expensive and difficult to apply
Coomassie stained
SDS-PAGE of
Affinity Chromatography
protein purification
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