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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
Keeping the Protein Native
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 - 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)
• Isoelectric Point – Proteins tend to be less soluble at the isoelectric point
• Storage– Proteins are not stable purified. Stored in glycerol or under gas to limit oxidation. -80oC limits
denaturing
How do you know?
How much of the protein being purified is in any sample during
purification? – Total protein assay doesn’t distinguish unless the
solution is pure… so what?
• If the protein being purified is an enzyme, conduct an enzyme activity
assay – based on the reaction catalyzed by the enzyme. Measure the gain
of product or loss of substrate (reactant).
• If the protein is not an enzyme (no measurable activity) use another
detection method – either a western blot or an ELISA assay (both antibody
based – more later this section)
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 organelles to isolate cell
fractions
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
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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)
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
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 non specific 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 S-transferase (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 Anion Exchange
•
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?
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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
Denatured Electrophoresis
- SDS PAGE
Separation of proteins based on size not charge – Denature by chemical and thermal means
Heat – defeats tertiary and quaternary features partially denatures protein
Reducing agent - ß-mercaptoethanol or dithiothreitol – Disulfide bonds
Detergent - sodium dodecyl sulfate (SDS)
- boil to unravel the protein and solvate protein
with ampipathic SDS
- each SDS has 2 negative charges
SDS-Protein 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
Immuno Analysis
Immunoglobins - 5 major classes main antibody in sera is IgG
•
antigen - foreign substance that triggers antibody formation
•
epitope - section of antigen that antibody recognizes
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
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Antibodies in specific analysis
ELISA (Enzyme Linked ImmunoAssay)- most sensitive detection methods for antibodies (aids test), proteins, peptides
and other substances (drug testing)
ELISA – Types
Three types of common ILISA All detect some form of antigen – protein or other
Direct – labeled primary antibody that reacts with the antigen
Indirect – Uses a secondary labeled antibody – most often used type of ELISA
Western blot - good for mixtures of proteins, identifying size and characteristics
– 1) transfer proteins form SDS PAGE to paper for antibody analysis.
– 2) Primary antibody recognizes protein antigen
– 3) secondary antibody recognizes the Fc region and is conjugated to a second
molecule to act as a signal
– 4) Enzyme linked to secondary generates a signal to be detected (light – Xray
film or camera, color ppt on paper)
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