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Transcript
Protein Purification and
Characterization
Why Study proteins?
IMPORTANT FACTORS IN PROTEIN
PURIFICATION
Starting materials
 tissues, cells or clones expressed in E. Coli or
animal cells
 Decisions– quantity of protein, protein modification
availability of samples, is it cloned yet,
expense
Stabilization of protein is key - proteins are not meant to be
purified, so you need to keep them alive and happy (active
/ native)
 pH - both activity and structure are pH dependent
 Temperature - most stabile at low temperature - reduces energy in
the system for unfolding and reduces the protease kinetics. Few
proteins are unstable at low temps - ppdk (Dr. Chastain's enzyme)
and the ATPase in mitochondria
 Protease inhibitors - several classes of proteins catalyze the
hydrolysis of peptide bonds (called proteases). Usually need to add
several "suicide" inhibitors and reduce free metals which are used
by the proteases
 Reducing agents - beta - mercaptoethanol and dithiolthreitol both
act as reducing agents. Prevent the oxidation of amino acids
 Detergents - Membrane bound proteins often need added
detergents (soaps) to mimic the ampipathic nature of the
membrane you so cruelly ripped it from - need to be above the
concentration at which micelles are formed - the critical micellular
concentration (CMC)
Homogenization - breaking the cell apart
Mechanical Shearing - Warring blender, glass or
plastic pestle like homogenizer
Freeze Thaw - cycles under hypnotic conditions,
ice crystals disrupts membranes
Enzymatic degradation of the cell
membrane - mostly for
bacterial preparation
Sonication - high energy sounds
to disrupt membrane
Detergent disruption of memb
METHODS OF PURIFICATION
Centrifugation - Separation based on density, mass, shape
and the density of the solution
 Sedimentation of particles measured by Svedburg units
 Force applied in gravitational (g) forces
 The centrifugal force depends on speed and time and
radius of rotor
 Differential centrifugation
 One of the most used methods in biochemistry
 Uses increasing g forces to yield a pellet and a
supernatant
 Subcellular centrifugation - a way to separate the cell
contents based on density of organelles
 Cytosol - not an organelle but a result of
centrifugation
Differential centrifugation
Use of density of organells to isolate cell fractions
Cyt osolic
fraction
600 g
x 10 min
Homogenat e
15,000 g
x 5 min
Nuclear
fraction
105,000 g
x 60 min
Mit ochondrial
fraction
Microsomal
fraction
Density gradient centrifugation
Also called Zonal centrifugation - Performed in the presence of an
increasing dense solution
often sucrose or other materials (percol most common)
can be used to purify a specific organelle or determine the
sedimentation and ultimately the molecular weight of a protein
Ammonium sulfate precipitation salting out proteins
At high concentrations of this strong salt, water is highly ordered
High concentration of strong chaotropic salts “strips” water away from
protein
Lower availability of solvent (water)
This favors protein interactions rather than protein - solvent interactions
causes aggregation of proteins (they become insoluble)
Each protein has a different
solubility so this is a
method to isolate groups
of protein
Precipitation is reversible and
usually non damaging to
structure of the enzyme
Ammonium Sulfate is most
commonly used. Urea is
also used but is usually is
harder for the protein to
refold.
Column Chromatography
Separation based the interactions between a mobile phase
and the chromatographic media (stationary phase)
Used to separate any of the big four biomolecules
Column Chromatography
Separation based the interactions between a mobile phase
and the chromatographic media (stationary phase)
Used to separate any of the big four biomolecules
Components
– Column
– Pump
– Absorbance monitor
– Conductivity monitor
– Fraction collector
– Controller - for more
advanced work
(control freaks?)
Affinity chromatography
purification based on a natural interactions for a protein and a substrate
or chemical group (ligand)
– only proteins which recognize the molecule on the stationary phase
will bind
– Elute by competition with the bound ligand
– generally a good method but it doesn’t always work - Some nonspecific interactions can occur
– Spacer arm may be needed to make the
compound available to the protein
Examples of ligand - protein
affinity
matrix
ATP. Glutathione, nickel – small molecules attached to a ligand
Fusion proteins can take advantage of affinity by acting
as a tag:
– glutathione S transferase (GST) –binds to glutathione
– histidine6 - binds to a nickel column
Power of biochemistry and molecular biology an example of affinity
chromatography
–
Ras - small protein involved in several cancers
 Low concentration in cells, so it difficult to purify and
study
 create a fusion protein 1/2 Ras 1/2 Glutathione Stransferase (GST) and produce large amounts of it.
 lead to discovery of additional proteins involved in Ras
regulation

Ion exchange chromatography
- separation of proteins based on net charge of protein - exchange of
ions for proteins
Anion Exchanger





weak exchanger - diethylaminoethyl (DEAE)
strong exchanger - quatenaryaminoethyl (QAE)
This type of resin is positively charged
The resin binds negative proteins
Proteins are eluted by NaCl or altering pH - how does this work?
Cation exchanger
 weak exchanger carboxymethy
(CM)
 weak exchanger sulfipropyl (SP)
 protein eluted by
the same means as
above
Size exclusion (SEC) or gel filtration chromatography
Media (solid phase) is a defined pore sizes in polymer beads,
large molecules “go around” small molecules “go through and around the
beads”
Smaller sized proteins are retained and come out last
Range of types of beads and chemistry - resin can be made of agarose,
acrylamide or other polymers
Size exclusion (SEC) or gel filtration chromatography
 Can be used to separate proteins, remove salts exchange buffers or to
determine the molecular weight of a purified protein
Also used to determine the molecular weight of a protein - use protein
standards with known molecular weights, prepare a standard curve of these
known proteins and compare the elution volumes of the knowns to the
unknowns
Example
 Sephacryl S-200 has a fractionation range of 5
kDa to 250 kDa
What is the exclusion limit?
– Would this be appropriate for a set of proteins
with molecular weights of 8 kDa, 15 kDa, 200
kDa and 500 kDa?
– What about 15, 250, 310, 405 kDa
– if you wanted the 15 kDa protein?
– What about if you wanted to purify the 310
kDa protein?
Protein Characterization
Electrophoresis - The transport of
particles by an electrical field through
a solid media
- a good method for determining the
purity of a protein and analyze a
mixture of proteins
- Separation of charged compounds
based on an applied electrical field, net
charge and frictional coefficient (mass
and shape of molecule)
Similar to DNA gels
- proteins and very small DNA
(oligonucleotides) use acrylamide
PAGE (Polyacrylamide Gel
Electrophoresis) the Gel is a
polymerized Acrylamide- alter the
ratio and concentration of polymer and
crosslinker to alter the pore size and
change the
migration through the gel
- A low % gel (acrylamide) will
separate higher MW proteins while
smaller proteins are not well resolved
-Stacking Gel vs. Resolving Gel - need
to get the proteins to start at the same
time (compressing the proteins into a
narrow starting zone at the resolving
gel)
Denatured Electrophoresis - SDS PAGE
Separation of proteins based on size not charge
Add a reducing agent - ß-mercaptoethanol or dithiothreitol
- and a detergent - sodium dodecyl sulfate (SDS)
- then boil to unravel the protein and solvate protein with
ampipathic SDS
- each SDS has 2 negative charges
- many SDS per molecule
- total amount of SDS bound is proportional to the MW
- Each protein molecule will be sufficiently negative
Therefore each protein will be very negativity charge regardless of the
amino acid composition,
- The size of protein influences the migration - separation is
based on size only not charge.
Native Gel Electrophoresis
Separations based on native size
and charge
Two proteins of a similar size but
different charge will migrate
differently
Protein interactions can influence
the migration of protein
Isoelectric Focusing Electrophoresis
– Separation of proteins based on isoelectric point
– Native or denatured electrophoresis in a pH gradient of
polyampholytes
– pH gradient is formed when electrical field is applie
– Proteins will migrate, depending on net charge, until there is no
longer a charge on the protein.
– How does this happen?
2 Dimensional Electrophoresis
Combination of native or denatured PAGE and IEF
Run in two directions
1- PAGE - to separate by size
2- IEF to separate by charge alone
Good to separate very crude mixtures or determine the
difference between two proteins that are the same size
but with a different pI
2D-Electrophoresis
2D-electrophoresis allows separation of
proteins by both size and isoelectric
point. Each spot represents a different
protein. The horizontal represents the
isoelectric focusing direction, while the
veritcal represents the SDS PAGE
direction.
Immuno Analysis
Immunoglobins - 5 major classes main antibody response in
sera is IgG
antigen - foreign substance that triggers antibody formation
epitope - section of antigen that antibody recognizes
Antibodies consist of
heavy and light chains
Fab region - highly
variable - recognize
target (antigen)
FC heavy chain - interacts
with other proteins
polyclonal vs. monoclonal
antibodies
polyclonal
– from sera of an animal
– several epitopes to the same antigen
– some may cross react with other proteins in a nonspecific manner
– produce lots of antibodies al long as the animal lives and
you continue to boost
monoclonal
– derived from single cell - hybrid of mouse spleen and a
immortal cell line (lymphocyte and myeloma)
– inject mice then can grow cell in a dish
– antibodies purified from cell culture media
– single epitope, very specific
– unlimited production of antibodies
Antibodies in specific analysis
ELISA (Enzyme Linked ImmunoAssay)- most sensitive
detection methods for antibodies (aids test),
proteins, peptides and other substances (drug
testing)
Plastic Dish
Antibodies in specific analysis
ELISA (Enzyme Linked ImmunoAssay)- most sensitive
detection methods for antibodies (aids test),
proteins, peptides and other substances (drug
testing)
1 Protein of interest is
Plastic Dish
Bound to plastic
Antibodies in specific analysis
ELISA (Enzyme Linked ImmunoAssay)- most sensitive
detection methods for antibodies (aids test),
proteins, peptides and other substances (drug
testing)
2 Unreacted binding sites are
Covered with a non-reactive
protein
1 Protein of interest is
Plastic Dish
Bound to plastic
Antibodies in specific analysis
ELISA (Enzyme Linked ImmunoAssay)- most sensitive
detection methods for antibodies (aids test),
proteins, peptides and other substances (drug
testing)
2 Unreacted binding sites are
3
Covered with a non-reactive
protein
Primary Antibody
Recognizes Antigen
1 Protein of interest is
Plastic Dish
Bound to plastic
Antibodies in specific analysis
ELISA (Enzyme Linked ImmunoAssay)- most sensitive
detection methods for antibodies (aids test),
proteins, peptides and other substances (drug
testing)
4
Secondary Antibody
Conjugated to an enzyme
3
2 Unreacted binding sites are
Covered with a non-reactive
protein
Primary Antibody
Recognizes Antigen
1 Protein of interest is
Plastic Dish
Bound to plastic
Antibodies in specific analysis
ELISA (Enzyme Linked ImmunoAssay)- most sensitive
detection methods for antibodies (aids test),
proteins, peptides and other substances (drug
testing)
5
4
Secondary Antibody
Conjugated to an enzyme
3
Enzyme reacts with
substrate producing
colored product
2 Unreacted binding sites are
Covered with a non-reactive
protein
Primary Antibody
Recognizes Antigen
1 Protein of interest is
Plastic Dish
Bound to plastic
Western blot - good for
mixtures of proteins,
identifying size and
characteristics
– transfer proteins form SDS
PAGE to paper for antibody
analysis.
– Primary antibody recognizes
protein antigen
– a secondary antibody
recognizes the Fc region and
is conjugated to a second
molecule to act as a signal