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Protein Purification and Characterization

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Why Study proteins? IMPORTANT FACTORS IN PROTEIN PURIFICATION Starting materials tissues, cells or clones expressed in E. Coli or animal cells Decisions- – PowerPoint PPT presentation

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Title: Protein Purification and Characterization


1
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

2
  • 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)

3
  • 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
4
  • 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

5
Differential centrifugation
  • Use of density of organells to isolate cell
    fractions

6
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

7
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.
8
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

9
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?)

10
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 doesnt always
    work - Some non-specific interactions can occur
  • Spacer arm may be needed to make the
  • compound available to the protein

11
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

12
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?

13
  • Cation exchanger
  • weak exchanger - carboxymethy (CM)
  • weak exchanger - sulfipropyl (SP)
  • protein eluted by the same means as above

14
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
15
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

16
  • 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?

17
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

18
  • 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)

19
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.

20
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

21
  • 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

22
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.
23
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
24
  • polyclonal
  • from sera of an animal
  • several epitopes to the same antigen
  • some may cross react with other proteins in a
    non-specific 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

25
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
26
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 Bound to plastic
Plastic Dish
27
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 Bound to plastic
Plastic Dish
28
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
3
Primary Antibody Recognizes Antigen
1
Protein of interest is Bound to plastic
Plastic Dish
29
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
2
Unreacted binding sites are Covered with a
non-reactive protein
3
Primary Antibody Recognizes Antigen
1
Protein of interest is Bound to plastic
Plastic Dish
30
Antibodies in specific analysis
  • ELISA (Enzyme Linked ImmunoAssay)- most sensitive
    detection methods for antibodies (aids test),
    proteins, peptides and other substances (drug
    testing)

5
Enzyme reacts with substrate producing colored
product
4
Secondary Antibody Conjugated to an enzyme
2
Unreacted binding sites are Covered with a
non-reactive protein
3
Primary Antibody Recognizes Antigen
1
Protein of interest is Bound to plastic
Plastic Dish
31
  • 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
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