Download BICH 605

BICH
605
Protein
Purification
1.
Sample Preparation
1.
2.
2.
Capture
1.
2.
3.
3.
Ion Exchange
Affinity
Hydrophobic Interaction
Intermediate Purification
1.
2.
4.
Extraction (grinding, detergent lysis, sonication)
Salt exchange (gel filtration, filters, dialysis)
Ion exchange
Hydrophobic Interaction
Polishing
1.
2.
Gel Filtration
Reversed phase
Protein Measurement: The Bradford Assay (Coomassie Blue Binding)
More Protein
Protein Measurement: The Bradford Assay (Coomassie Blue Binding)
Build a Standard Curve
Protein Variation is Great
Protein Measurement: The Bicinchoninic Acid (BCA) Assay
CAPTURE METHODS
• ION EXCHANGE
• HYDROPHOBIC INTERACTION
• AFFINITY CAPTURE
ION EXCHANGE CHROMATOGRAPHY
•
ION EXCHANGE BINDING IS
BASED ON SURFACE CHARGES
OF PROTEINS
•
PROTEINS ARE IONS
•
ELUTION IS CAUSED BY
COMPETITION WITH COUNTER
IONS IN BUFFER (OFTEN SODIUM
CHLORIDE)
•
CHARGE IS A FUNCTION OF pH
pI = ISOLECTRIC POINT
BELOW pI NET CHARGE
IS POSITIVE (a cation)
ABOVE pI NET CHARGE
IS NEGATIVE (an anion)
ION EXCHANGE CHROMATOGRAPHY
An example of Ion Exchange
Protein acts like a CATION
Protein acts like an ANION
“Exchange” protein cation with
a Cationic Counterion
ION EXCHANGE CHROMATOGRAPHY
•
IEX CAN BE EITHER ANION
EXCHANGE OR CATION
EXCHANGE
•
MANY DIFFERENT MATRICES
AVAILABLE COMMERCIALLY
•
CHOOSE BASED ON
STABILITY OF PROTEIN AT
pH OF OPERATION AND
PROPERTIES OF ‘OTHER’
PROTEINS IN MIXTURE
•
STRONG AND WEAK
BINDERS CHARACTERIZE
BINDING STRENGTH
ION EXCHANGE CHROMATOGRAPHY
SET UP OF APPARATUS
COMPONENTS:
•BUFFER RESERVOIRS
•COLUMN FILLED WITH MATRIX
•MONITOR & RECORDER (not shown)
•FRACTION COLLECTOR (not shown)
ION EXCHANGE CHROMATOGRAPHY
GRADIENT ELUTION TECHNIQUES
HIGH SALT
LOW SALT
Linear Gradient
Step Gradient
Detector
Fraction
Collector
animation
ION EXCHANGE CHROMATOGRAPHY
FLOWRATE AND GRADIENT
SLOPE EFFECT RESOLUTION
Generally achieve 5-10 fold
enrichment of target proteins
depending upon sample complexity
and steepness of gradient. Often
used as a first chromatographic
step in purification as it can
removed oppositely charged
molecules immediately. Useful in
presence of non-ionic detergents,
urea and other chaotropes. Scaling
options are wide. Fractions can be
applied directly to hydrophobic
interaction chromatography
matrices (high salt). A
“concentrating” method.
Shallow Gradient; 8 ml/hr
Steep Gradient; 8 ml/hr
Shallow Gradient; 20 ml/hr
CAPTURE METHODS
• ION EXCHANGE
• HYDROPHOBIC INTERACTION
• AFFINITY CAPTURE
HYDROPHOBIC INTERACTION
CHROMATOGRAPHY (HIC)
HIC SEPARATES BIOMOLECULES BASED ON THE HYDROPHOBIC GROUPS ON THEIR
SURFACES.
Binding of biomolecules to the mildly
hydrophobic surface of a HIC column is
induced by the addition of high salt
concentrations to the sample and
equilibration buffer. Elution is effected
by decreasing the salt concentration.
HYDROPHOBIC INTERACTION CHROMATOGRAPHY (HIC)
ELUTION FROM HIC COLUMN IS BY
DECREASING IONIC STRENGTH OF BUFFER
(Note: This is OPPOSITE of Ion Exchange
Chromatography).
DIFFERENT SALTS MAY BE USED;
AMMONIUM SULFATE IS MOST COMMONLY
USED
HYDROPHOBIC INTERACTION CHROMATOGRAPHY (HIC)
DIFFERENT FUNCTIONAL GROUPS ARE
AVAILABLE WITH DIFFERENT DEGREES OF
HYDROPHOBICITY
HYDROPHOBIC INTERACTION CHROMATOGRAPHY (HIC)
DIFFERENT FUNCTIONAL GROUPS HAVE
DIFFERENT SELECTIVITY
Cannot be used with
detergent containing
buffers (hydrophobic
column binds detergents
to exclusion of proteins).
Useful as a preceding
step to IEX as it is eluted
in low salt buffer. A
“concentrating” method.
CAPTURE METHODS
• ION EXCHANGE
• HYDROPHOBIC INTERACTION
•
AFFINITY CAPTURE
AFFINITY CHROMATOGRAPHY
A TYPE OF ADSORPTION
CHROMATOGRAPHY IN WHICH
THE MOLECULE TO BE
PURIFIED IS SPECIFICALLY
AND REVERSIBLY ADSORBED
BY A COMPLEMENTARY
BINDING SUBSTANCE (LIGAND)
IMMOBILIZED ON AN
INSOLUBLE SUPPORT
(MATRIX).
PURIFICATION IS OFTEN OF
THE ORDER OF SEVERAL
THOUSAND FOLD; RECOVERY
OF ACTIVE MATERIAL CAN BE
VERY HIGH.
AFFINITY CHROMATOGRAPHY
HOW TO DESIGN AN AFFINITY MATRIX
ANY COMPONENT CAN BE USED
AS A LIGAND. IT IS IMPORTANT
THAT THE IMMOBILIZED LIGAND
RETAINS ITS SPECIFIC BINDING
ACTIVITY.
CORRECT CHOICE OF COUPLING
GEL IS DICTATED BY BOTH THE
TYPE OF GROUPS AVAILABLE ON
THE LIGAND MOLECULE FOR
COUPLING AND THE NATURE OF
THE BINDING REACTION.
IMMOBILIZATION SHOULD BE
ATTEMPTED THROUGH THE
LEAST CRITICAL REGION OF THE
LIGAND.
SPACER ARMS?
AFFINITY CHROMATOGRAPHY
COUPLING REACTIONS WITH ACTIVATED
RESINS
AFFIGEL RESINS (BIORAD)
Aqueous buffer, pH 3-10
N-hydroxysuccinimide
ester coupling reaction for
ligands with primary
amines
CYANOGEN BROMIDE-ACTIVATED
RESIN (NO SPACER)
Aqueous buffer, pH 8-10
(more acidic is slightly less
efficient, but slower
spontaneous hydrolysis of
ester) for ligands with
primary amines
IMMOBILIZED METAL AFFINITY
CHROMATOGRAPHY (IMAC)
Immobilized metal affinity
chromatography (IMAC), proteins and
peptides separates on the basis of
their affinity for metal ions which have
been immobilized by chelation.
Histidine and cysteine form
complexes with the chelated
metals around neutral pH
(pH 6-8).
Transitional metals are most
often used for IMAC.
IMMOBILIZED METAL AFFINITY CHROMATOGRAPHY (IMAC)
Binding is pH dependent and elution is
carried out by reducing the pH or
increasing ionic strength of the buffer.
Another widely used elution method is to
use EDTA in the buffer.
This method is very useful and widely
used to purify recombinant proteins that
have been made as over-expressed, Histag fusion proteins.
IMMOBILIZED METAL AFFINITY CHROMATOGRAPHY (IMAC)
NICKEL IMAC HIS-TAG PURIFICATION
SDS PAGE
POLISHING METHODS
• GEL FILTRATION
• REVERSED PHASE HPLC
Gel Filtration Chromatrography (GFC)
(Size Exclusion Chromatrography)
Principle
•
Gel filtration is performed using
porous beads as the
chromatographic support. GFC
separates proteins based on
size.
•
Separation is accomplished by
the differential in ‘residence
time’ a protein spends inside of
or outside of the porous beads.
Gel Filtration Chromatrography
The elution volume (Ve) of a protein can be
calculated as a partition coefficient (Kav)
using the following equation where Vt
equals the sum of the external volume
and the internal volume:
A semi-logarithmic plot of the dependence of
the partition coefficient (Kav) on molecular
weight is illustrated here. The
separation of proteins based on
molecular weight will be greatest in
the central linear region of this
sigmoidal relationship, spanning Kav
values between 0.2 and 0.8. This span is
described as the fractionation range of
a size-exclusion matrix.
Gel Filtration Chromatrography
GEL FILTRATION MATRICES ARE AVAILABLE TO SEPARATE A WIDE RANGE OF
MOLECULAR SIZES AND CONSTRUCTED FROM A VARIETY OF MATERIALS
SEPHADEX™ Cross-linked dextrans
(range 1-600 kDa)
SUPERDEX™ Dextran/agarose matrix
(range 3-600 kDa)
SEPHAROSE™
(range 10-40,000 kDa)
Gel Filtration Chromatrography
GENERALIZED GFC SETUP
animation
Gel Filtration Chromatrography
GFC IS USEFUL FOR DESALTING
PROTEIN SOLUTIONS (vs.
DIALYSIS)
Gel Filtration Chromatrography
EFFECT OF FLOWRATE AND BED HEIGHT ON RESOLUTION AND
GEL FILTRATION CHROMATOGRAPHY
•
1 vs 2 EFFECT OF FLOWRATE
•
2 vs 3 EFFECT OF BED HEIGHT
Generally, fewer than 10 proteins can be
resolved from one another in the effluent of a
size exclusion column. This being the case, it
is wise to perform this method relatively
late in a purification procedure when the
numbers of proteins are relatively small and
when the preceding step has fractionated the
protein mixture on the basis of a completely
different property.
1
2
3
END OF DAY 2