Protein Purification

1
Protein Purification

• Before any particular protein can be sequenced
  and characterized, its is necessary to separate
  it from all of the other proteins in the cell.
• This purification process involves separating
  proteins based on their ionic properties, their
  sizes, their hydrophobicity, and their affinities
  for certain molecules (ligands). Each successive
  step is referred to as fractionation.
• Typically some form of column chromatography is
  employed, in which the solid phase (stationary
  phase) contains molecules that in some way
  exploit the differences among various proteins.

2
Ion-Exchange Chromatography

• This procedure is very similar to the procedure
  that we talked about for separating amino acids
  based on charge.
• The picture at the left represents ion-exchange
  chromatography on a cation exchanger. Notice
  that the bead is negatively charged, and
  therefore the rate of mobility of proteins loaded
  onto the resin is proportional to the degree of
  negative charge that they bear.

3
Size Exclusion Chromatography

• In size-exclusion chromatography
  (gel-filtration chromatography), proteins migrate
  as a function of their molecular weights.
• The solid matrix (beads) contains pores of
  various sizes. The probability of entering the
  pores of the matrix is inversely proportional to
  the size of the protein. In fact proteins that
  are larger than a given size (depending on the
  resin that is used) are totally excluded from
  entering the beads. Therefore, larger proteins
  have a more direct route to the bottom of the
  column, by simply going around all of the beads
  rather than entering the beads.
• Notice in the figure at left that the large
  molecules elute first.
• Size-exclusion chromatography can be used to
  determine the molecular weight of a particular
  protein if appropriate standards are available.
  As well see in a couple of slides, the elution
  volume is inversely proportional to the log of
  the molecular weight.

4
Affinity Chromatrography

• In affinity chromatography, proteins are
  separated according to their ability to bind to a
  specific ligand that is connected to the beads of
  the resin.
• After the proteins that do not bind the ligand
  are washed through the column, the bound protein
  of interest is eluted by a solution containing
  free ligand.

5
Salting Out

• The first step in purifying a protein is
  establishing a crude extract. This requires
  that the membrane of the cell be ruptured by some
  technique.
• Upon rupturing the membrane and releasing the
  contents of the cell, the insoluble debris is
  removed by centrifugation.
• Typically, one of the initial steps involves
  purifying proteins based on their solubilties in
  varying concentrations of ammonium sulfate. The
  solubility of a protein is sensitive to the
  concentrations of dissolved salts. The
  solubility of a protein at low ionic strength
  generally increases with the salt concentration
  (salting in). At high ionic strength, the
  solubilities of proteins decreases (salting out).
• Many unwanted proteins can be eliminated by
  adjusting the salt concentration in a solution
  containing the crude extract to just below the
  precipitation point of the protein to be
  purified. In this case the protein to be
  purified remains in solution, while many others
  are precipitated. Likewise, unwanted proteins
  can be eliminated by adjusting the salt
  concentration to just over the precipitation
  point of the protein of interest. In this case,
  the protein of interest will be precipitated
  while many others will remain in solution.

6
Specific Activity

• In order to purify a protein or any other
  substance, there must be a means of
  quantitatively detecting its presence.
• Therefore, an assay must be determined.
  Typically assays are based on the function of a
  protein. For example, a substrate like shown
  below might be used for an enzyme that cleaves an
  ester bond (esterase). One of the products of
  this reaction has an intense absorption at 412
  nm, which would allow for the detection of the
  enzyme.
• You would be interested in how fast the enzyme
  cleaves this molecule (rate of color
  development), and how much protein is being added
  to the reaction mixture. Your specific activity
  would be the rate divided by the total amount of
  protein. The higher the specific activity the
  more pure your protein.

Intense absorption at 412 nm (yellow)
Esterase

7
Hypothetical Purification
8
Electrophoresis
Electrophoresis is based on the migration of
proteins in a charged field. The force moving
the macromolecules is the electrical potential,
E. µ V/E, where µ is the electrophoretic
mobility, and V is the velocity of the
particle. µ Z/f, where Z is the net charge of
the molecule, and f is the frictional
coefficient. f is related to the the shape of the
molecule, as well as its size. Typically, the
cross-linked polymer, polyacrylamide acts as the
solid support. Normally, proteins would be
separated in proportion to their charge-to mass
ratio. The problem is that some proteins would
migrate towards the anode, while others would
migrate toward the cathode and, the migration
would not reflect size, but charge to mass. The
trick is to carry out the electrophoresis in the
presence of sodium docecyl sulfate, which is a
detergent. SDS binds to every protein in roughly
the same proportion, which is about one molecule
for every two amino acid residues. SDS carries
with it a negative charge, and the cumulative
negative charge renders the intrinsic net charge
of the protein insiginificant. Therefore, every
protein will have the same charge to mass ratio,
which will cause all proteins to migrate towards
the cathode with a rathe that is dependent on
their sizes. In contrast to gel-filtration,
smaller molecules migrate faster than larger
molecules Typically, this technique is not used
to purify proteins, because SDS normally
denatures proteins. It is used to analyze the
purity of proteins.
9
Molecular Weight Determination
Shown above (on the left) is an
SDS-polyacrylamide gel that has been stained with
a dye in order to view the proteins. Lane one of
the left figure represents a set of standards,
while lane 2 represents a protein that has been
purified. The molecular weight Mr of the protein
can be estimated, by comparing its relative
migration to that of the standards. The relation
is that the relative migration is dependent on
the log of the molecular weight.
10
Isoelectric Focusing
Isoelectric focusing is a procedure that will
allow the pI of a particular protein to be
determined, and that will separate proteins based
on their respective pIs. In this technique, a
mixture of proteins is applied to a gel that
contains a pH gradient. At the point along the
gradient wherein the protein is no longer
charged, it will cease to migrate. This point is
the pI of the protein, or the isoelectric point.
11
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.
12
What Can You Get from Sequence Information?
13
Homologous Proteins are Related by Primary
Sequence

• Homologous proteins are proteins from different
  species that are evolutionarily related as
  determined by their primary structures (sequence
  of amino acids).
• Proteins that are highly homologous usually
  perform the same reaction in the corresponding
  organisms, and usually have very similar
  three-dimensional structures.
• Homologous proteins from different species may
  have polypeptide chains that are identical or
  nearly identical in length.
• Many positions in the amino acid sequence are
  occupied by the same residue in all species.
  These are called invariant residues.
• Positions that show considerable variation in the
  amino acid residue from one species to another
  are called variable residues.
• Below is a pseudo amino acid sequence alignment
  of the protein cytochrome C. The top line
  represents the human protein. Amino acids in
  yellow are those that are invariant across
  species. Those in blue indicate conservative
  substitutions.

14
Lipoyl Synthase Sequence Alignment
Motif III
Motif II
Motif I
CYRXCXFCXV
TKXXXMXGXGE
VCXEAXCXNXXEC



15
Protein Homology Among Species

• Variable amino acids can provide information
  concerning the phylogenetic (evolutionary)
  relationships among various species.
• The number of residues that differ in homologous
  proteins from any two species is in proportion to
  the phylogenetic difference between those
  species.
• 48 amino acid residues are variable between
  cytochromes C from the horse and yeast. In
  contrast, only two residues differ between the
  cytochromes C of duck and chicken.
• Within humans, 20 to 30 of the proteins are
  considered to be polymorphic, which means that
  they have amino acid sequence variations that do
  not result in loss of function of the protein.

16
Relevant and Important Websites

• There are a number of website that will allow you
  to obtain a wealth of information about a
  protein, it the primary sequence is known, or if
  the DNA sequence is known.
• Additionally, in contrast to the laborious
  methods associated with sequence determination,
  since the entire genomes of many organisms have
  already been sequenced, a small amount of peptide
  information will allow the identity of a protein
  to be determined.
• http//us.expasy.org/
• http//us.expasy.org/alinks.htmlProteins
• http//us.expasy.org/tools/