Lecture 7: Protein purification

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Lecture 7 Protein purification

• Protein purification

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Methods of solubilization

• 1st step is to get the protein into solution
• If the protein is located in the cytosol, we need
  to break open the cell.
• For animal cells this can be accomplished with
  osmotic lysis. Put the cells in hypotonic
  solution less solutes than inside the cell.
• For cells with a cell wall (bacteria, plants) we
  have to use other methods.
• For bacteria, lysozyme is often effective -
  Selectively degrades bacterial cell wall.
• Can also use detergents or organic solvents,
  although these may also denature the protein.

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Mechanical processes to break open the cell

• High speed blender
• Homogenzier
• French press
• Sonicator.
• After the cells have been broken, the crude
  lysate, may be filtered or centrifuged to remove
  the particulate cell debris. The protein of
  interest is in the supernatant.

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For proteins that are components of membranes or
subcellular assembly

• Remove the assembly from the rest of the cellular
  material (mitochondria for example).
• Can be done by differential centrifugation-cell
  lysate is centrifuged at a speed that removes
  only the cell components denser than the desired
  organelle followed by a centrifugation at speed
  that spins down the organelle.

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Stabilization of proteins

• pH
• Temperature
• Inhibition of proteases
• Retardation of microbes that can destroy proteins
• Sodium azide is often used.

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Assay of proteins

• Done throughout the purification process to make
  sure that your protein of interest is there.
• If the protein of interest is an enzyme, using a
  reaction for which that enzyme is a catalyst is a
  good way to monitor protein recovery.
• Monitor the increase of the product of the
  enzymatic reaction
• Fluorescence
• Generation of acid to be monitored by titration
• Coupled enzymatic reaction - couple with another
  enzyme to make an observable substance.

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Immunochemical techniques to assay for proteins

• Use specific immunoglobulins (antibodies),
  proteins that interact specifically with the
  protein of interest and can be easily monitored.
• Antibodies are produced by an animals immune
  system in response to the introduction of a
  foreign protein.
• Antibodies specifically bind to the foreign
  protein.
• Extracted from blood serum of animal that has
  been immunized against a particular protein.
• Many different antibodies in sera with different
  specificities and binding affinities toward the
  protein of interest.

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Immunochemical techniques to assay for proteins

• Immune cells that produce antibodies normally die
  after a few cell divisions so it is difficult to
  get a specific antibody clone.
• We can make monoclonal antibodies by fusing a
  cell producing the desired antibody with a cancer
  cell (myeloma).
• This results in a hybridoma that is essentially
  an immortal cell, so large quantities of
  monoclonal antibody can be produced.

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Figure 6-1 An enzyme-linked immunosorbent assay
(ELISA).
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Summary of initial steps of protein purification

• Choose source of proteins.
• Solubilize proteins.
• Stabilize proteins.
• Specific assay for protein of interest
• Enzymatic activity, immunological activity,
  physical characteristics (e.g. molecular mass,
  spectroscopic properties, etc.), biological
  activity
• Assay should be
• Specific
• Rapid
• Sensitive
• Quantitative

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Things to monitor during protein purification

• Things to monitor during protein purification
• Total sample volume
• Total sample protein (est. by A280 1.4-1.0
  mg/ml)
• Units of activity of desired protein (based on
  specific assay)
• Other basic information to track
• yield for each purification step
• Specific activity of the desired protein
  (units/mg of protein)
• Purification enhancement of each step (e.g. 3.5
  fold purification)
• In designing a purification scheme you have to
  balance purification with yield.

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Solubilities of proteins

• Multiple acid-base groups on proteins affect
  their solubility properties.
• Solubility of a protein is therefore dependent on
  concentrations of dissolved salts, the polarity
  of solvent, the pH and the temperature.
• Certain proteins will precipitate from solutions
  under conditions which others remain soluble-so
  we can use this as an initial purification step
  of proteins.
• Salting out or salting in procedures take
  advantage of ionic strength

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1/2?ciZi
Ionic strength (I)
Ci molar concentration of ionic species Zi
ionic charge
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Figure 6-2 Solubilities of several proteins in
ammonium sulfate solutions.
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Solubilities of proteins

• A proteins solubility at a given ionic strength
  varies with the types of ions in solution.
• The order of effectiveness of these various ions
  in influencing protein solubility is quite
  similar for different proteins and is due to the
  ions size and hydration.
• The solubility of a protein at low ionic strength
  generally increases with the salt concentration.
  This is called salting in. As the salt
  concentration increases the additional
  counterions more effectively shield the protein
  molecules multiple ionic charges and thereby
  increase the proteins solubility.
• At high ionic strengths the solubilities of
  proteins as well as most other substances
  decrease. This is called salting out and results
  from a competition between the added salt ions
  and the other dissolved solutes for molecules of
  solvation.

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Figure 6-3 Solubility of caboxy-hemoglobin at its
isoelectric point as a function of ionic strength
and ion type.
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Solubilities of proteins

• Salting out is one of the most commonly used
  protein purification procedures.
• By adjusting the salt concentration in a solution
  with a mixture of proteins to just below the
  precipitation point of the protein to be
  purified, many unwanted proteins can be
  eliminated from solution. Then after the
  precipitate is removed by filtration or
  centrifugation, the salt concentration of the
  remaining solution is increased to precipitate
  the desired protein.
• Ammonium sulfate is the most commonly used
  reagent
• High solubility (3.9 M in water at 0 ºC)
• High ionic strength solution can be made (up to
  23.5 in water at 0 ºC)

Note-certain ions (I-, ClO4-, SCN-, Li, Mg2,
Ca2 and Ba) increase the solubilities of
proteins rather than salting out. (also denature
proteins).
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Solubilities of proteins

• Water-miscible organic solvents also precipitate
  proteins.
• Acetone, ethanol
• Low dielectric constants lower the solvating
  power of their aqueouse solutions for dissolved
  ions.
• This technique is done at low temperatures (0 ºC)
  because at higher temperatures, the solvent
  evaporates.
• Can magnify the differences in salting out
  procedures.
• Some water-miscible organic solvents (DMF, DMSO)
  are good at solubilizing proteins (high
  dielectric constants).

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Solubilities of proteins

• Proteins have various ionizable groups (many
  pKs)
• At a pH characteristic for each protein, the
  positive charges on the molecule exactly balance
  the negative charges (isoelectric point, pI).
• At pI, the protein has no net charge and is
  immobile in an electric field.
• Therefore, solubility can be influenced by
  changes in the pH.

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Figure 6-4 Solubility of b-lactoglobin as a
function of pH at several NaCl concentrations.
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Solubilities of proteins

• A protein in a pH near its isolectric point is
  not subject to salting in.
• As the pH is moved away from the pI of the
  protein, the proteins net charge increases and
  it is easier to salt in.
• Salts inhibit interactions between neighboring
  molecules in the protein that promote aggregation
  and precipitation.
• pIs of proteins can be used to precipitate
  proteins.

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Table 6-1 Isoelectric Points of Several Common
Proteins.
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Column chromatography

• After the initial fractionation steps we move to
  column chromatography.
• The mixture of substances (proteins) to be
  fractionated is dissolved in a liquid or gaseous
  fluid called the mobile phase.
• This solution is passed through a column
  consisting of a porous solid matrix called the
  stationary phase. These are sometimes called
  resins when used in liquid chromatography.
• The stationary phase has certain physical and
  chemical characteristics that allow it to
  interact in various ways with different proteins.
• Common types of chromatographic stationary phases
• Ion exchange
• Hydrophobic
• Gel filtration
• Affinity

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Ion exchange chromatography

• Ion exchange resins contain charged groups.
• If these groups are acidic in nature they
  interact with positively charged proteins and are
  called cation exchangers.
• If these groups are basic in nature, they
  interact with negatively charged molecules and
  are called anion exchangers.

Positively charged (basic) protein or enzyme

CH2-COO-

CH2-COO-


CM cellulose cation exchanger
CH2-CH2 -NH(CH2CH2)
CH2-CH2 -NH(CH2CH2)
DEAE cellulose anion exchanger
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Ion exchange chromatography
For protein binding, the pH is fixed (usually
near neutral) under low salt conditions. Example
cation exchange column
Positively charged protein or enzyme bind to the
column




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Ion exchange chromatography
To elute our protein of interest, add
increasingly higher amount of salt (increase the
ionic strength). Na will interact with the
cation resin and Cl- will interact with our
positively charged protein to elute off the
column.
Increasing NaCl of the elution buffer
Cl-
Na
Na2
Cl-
Na
Cl-
Cl-
Na2
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Ion exchange chromatography

• Proteins will bind to an ion exchanger with
  different affinities.
• As the column is washed with buffer, those
  proteins relatively low affinities for the ion
  exchange resin will move through the column
  faster than the proteins that bind to the column.
• The greater the binding affinity of a protein for
  the ion exchange column, the more it will be
  slowed in eluting off the column.
• Proteins can be eluted by changing the elution
  buffer to one with a higher salt concentration
  and/or a different pH (stepwise elution or
  gradient elution).
• Cation exchangers bind to proteins with positive
  charges.
• Anion exchangers bind to proteins with negative
  charges.

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Figure 6-6 Ion exchange chromatography using
stepwise elution.
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Ion exchange chromatography

• Gradient elution can improve the washing of ion
  exchange columns.
• The salt concentration and/or pH is continuously
  varied as the column is eluted so as to release
  sequentially the proteins bound to the column.
• The most widely used gradient is the linear
  gradient where the concentration of eluant
  solution varies linearly with the volume of the
  solution passed.
• The solute concentration, c, is expressed as
• c c2 - (c2 - c1)f
• c1 the initial concentration of the solution in
  the mixing chamber
• c2 the concentration of the reservoir chamber
• f the remaining fraction of the combined
  volumes of the solutions initially present in
  both reservoirs.

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Figure 6-7 Device for generating a linear
concentration gradient.
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c c2 - (c2 - c1)f
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Figure 6-8 Molecular formulas of cellulose-based
ion exchangers.
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Table 6-2 Some Biochemically Useful Ion
Exchangers.
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Ion exchange chromatography

• Ion exchangers can be cellulosic ion exchangers
  and gel-type ion exchangers.
• Cellulosic ion exchangers most common.
• Gel-type ion exchangers can combine with gel
  filtration properties and have higher capacity.
• Disadvantage-these materials are easily
  compressed so eluant flow is low.
• There are other materials derived from silica or
  coated glass beads that address this problem.

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Gel filtration chromatography

• Also called size exclusion chromatography or
  molecular sieve chromatography.
• How does it work? If we assume proteins are
  spherical

Molecular mass (daltons) 10,000 30,000 100,0
00
size
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Gel filtration chromatography
flow
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Gel filtration chromatography
flow
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Gel filtration chromatography
flow
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Gel filtration chromatography
flow
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Gel filtration chromatography
flow
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Gel filtration chromatography

• The molecular mass of the smallest molecule
  unable to penetrate the pores of the gel is at
  the exclusion limit.
• The exclusion limit is a function of molecular
  shape, since elongated molecules are less likely
  to penetrate a gel pore than other shapes.
• Behavior of the molecule on the gel can be
  quantitatively characterized.

Total bed volume of the column Vt Vx V0
Vx volume occupied by gel beads
V0 volume of solvent space surrounding gel
Typically 35
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Gel filtration chromatography

• Elution volume (Ve) is the volume of a solvent
  required to elute a given solute from the column
  after it has first contacted the gel.
• Relative elution volume (Ve/V0) is the behavior
  of a particular solute on a given gel that is
  independent of the size of the column.
• This effectually means that molecules with
  molecular masses ranging below the exclusion
  limit of a gel will elute from a gel in the order
  of their molecular masses with the largest
  eluting first.

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Figure 6-9 Gel filtration chromatography.
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Figure 6-10 Molecular mass determination by gel
filtration chromatography.
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Table 6-3 Some Commonly Used Gel Filtration
Materials.
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Gel filtration chromatography

• Elution volume (Ve) is the volume of a solvent
  required to elute a given solute from the column
  after it has first contacted the gel.
• Relative elution volume (Ve/V0) is the behavior
  of a particular solute on a given gel that is
  independent of the size of the column.
• This effectually means that molecules with
  molecular masses ranging below the exclusion
  limit of a gel will elute from a gel in the order
  of their molecular masses with the largest
  eluting first.

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Affinity chromatography

• Many proteins can bind specific molecules very
  tightly but noncovalently.
• We can use this to our advantage with affinity
  chromatography.

Glucose (small dark blue molecule) binding to
hexokinase. The enzyme acts like a jaw and
clamps down on the substrate (glucose)
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Affinity chromatography

• How does it work?
• Ligand - a molecule that specifically binds to
  the protein of interest.

Inert support


Ligand
Spacer arms
Affinity material prepared
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Affinity chromatography
Mixture of proteins
Unwanted proteins
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Affinity chromatography
Elute with competitive ligand.
Inert support
Remove from competitive ligand by dialysis.
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Affinity chromatography

• To remove the protein of interest from the
  column, you can elute with a solution of a
  compound with higher affinity than the ligand
  (competitive)
• You can change the pH, ionic strength and/or
  temperature so that the protein-ligand complex is
  no longer stable.

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Immunoaffinity chromatography

• Monoclonal antibodies can be attached to the
  column material.
• The column only binds the protein against which
  the antibody has been raised.
• 10,000-fold purification in a single step!
• Disadvantges
• Difficult to produce monoclonal antibodies
  (expensive !)
• Harsh conditions to elute the bound protein