Proteins purification

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Protein Purification
M.Prasad Naidu MSc Medical Biochemistry, Ph.D,.
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Protein Isolation

• Must have sensitive method for detection.
• Select a good source for the protein.
• a. Rich source of material.
• i.e. Heart mitochondria for cytochrome C
• b. bakers yeast (Saccharomyces cerevisiae)
• c. Escherichia coli (recombinant expression)
• Tissue specificity Brain vs. kidney vs. eye.
• Chickens, cows, pigs or rats are often used.
• Molecular cloning techniques have allowed
  biochemists to over-express desired proteins in
  bacteria or C.H.O. (Chinese Hamster Ovary) cells
  by isolating the gene and placing it into a host
  system.

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Methods of solubilization animal cells

• Cells can be lysed by hypotonic shock.
• Cells with high salt inside and no salt outside
  will
• swell and rupture
• Bacteria outer membranes must be digested.
• Gram-negative bacteria
• Hen egg white lysozyme digests b (1-4) linkages
  in the (glycosidic bonds) of polysaccharides.
• Mechanical breakage blenders homogenizers
• French press - high pressure 20,000 lbs/in2
  forced through a small hole disrupts cells
• ultrasound or sonication disrupts cells.

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• Centrifugation
• Lysate - broken (lysed) cells- can be separated
  using
• differential centrifugation
• ? RPM - spun down
• separates by density differences or by size (MW)
  of particles.
• Cellular fractionation can separate
• mitochondria
• microsomes
• ribosomes
• soluble proteins

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Centrifugation Units
Where w angular velocity v velocity of
particle R distance from center of rotation M
molecular weight V partial specific volume of
particle r density of solvent Sedimentation
velocity (Svedberg Coefficient) S s x 10-13
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Stability proteins can denature!!
H-bonds, ionic bonds, Van der Waals interactions,
and Hydrophobic interactions can be disrupted.
Denaturation is the process by which a protein
loses its native or active shape or
conformation. Temperature can play a role cold
labile heat labile Protect against-Proteases,
Inhibitors, Changes in pH, Protein can be
air-denatured -egg white meringue - absorption to
surfaces Damaged by oxidation 02 Heavy and
transition metals damage proteins -they bind to
protein- Cu Hg Bacterial contamination can
destroy the protein
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Activity Measurements
In order to follow the purity of an enzyme, you
need a method to measure its activity. Spectrapho
tometric analysis- is one common method to
measure activity. Substrate S
Product P a change of S with time if S is
colored absorbs light we can use Beers Law. A
eb c c - concentration e - millimolar
extinction coefficient A - absorbance b - path
length T - percent transmittance
A - log T
if ? A then ? c at ? max
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enzyme
For the reaction NADH ? NAD H-
NADH
l Max 340 nm
DA
Absorbance
NAD
300 nm
350 nm
Volume is 1 ml so micromoles NADH oxidized

Specific activity
DT min
mg
mg of protein
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Start with one liter of lysed cells. We measure
the rate of .01 ml of cells at at concentration
of 20 mg/ml. i.e. the amount of enzyme we will
assay is 0.01 ml We get a rate of ? A 0.5
A/min 1 millimolar 6.22 DA e mM 0.5/6.22
.008 millmolar/min and our assay volume 1
ml 1 millimolar in a volume of one ml 1
micromole/ml ?mole ?C.008 ?moles in 1 ml/min
.04 ?moles 0.2 mg min/mg
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Total activity .04 mmoles x 20 mg/ml 0.8
mmoles / ml 0.8 ?moles x 1000 ml 800 ?moles
in 1 liter of cells ml
min Red is our enzyme If we remove
greens blues the specific activity increases,
however, our total activity remains the same. If
We lose red the total activity decreases.
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We usually monitor both the total activity and
specific activity for each purification
step. Until the Specific Activity reaches a
maximal value. How do we know if it is pure?
Usually SDS - Page See Table 5-4 in Voet and
Voet Some enzymes have no easy assay but the
product of the reaction can be used in another
reaction enz1 enz2 A B
C NADH
NAD Coupled Reactions We couple enz2 to enz1
and measure ?NADH to get ?A
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Use of radioactivity
ATP ? ADP Pi Separate ATP Pi ADP on
TLC measure radioactivity Phosphoimager
makes this easy else cut spots and count in
scintillation counter.
Pi
ATP
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Strategy of Purification

• Fractionation procedures or steps to isolate
  protein based on physical characteristics.
• Characteristic
  Procedure
• Charge 1. Ion exchange
• 2. Electrophoresis
• 3. Isoelectric focusing
• Polarity 1. Adsorption
  chromatography
• 2. Paper chromatography
• 3. Reverse phase chromatography
• 4. Hydrophobic interaction

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• Characteristic
  Procedure
• Size 1. Dialysis and ultrafiltration
• 2. Gel electrophoresis
• 3. Gel filtration
• 4. Ultracentrifugation
• Specificity 1. Affinity chromatography
• 2. Immunopurification
• Solubility 1. Salt precipitation
• 2. Detergent solubilization

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Ionic Strength
Ci the molar concentration of the ith
species Zi its ionic charge 1M Na Cl- Z
1 Na Z 1 Cl- 1
(1M x 1)Na (1M x 1)Cl 2
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For di- or tri-valent ions, where I is different
than M 1M MgCl2 Mg 1M, and Z
2 while Cl- 2M, and Z 1 I (1 x 22)Mg
(2 x 12)Cl 4 2 3 2 2
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Salting out
Use (NH4)2 SO4 it is a Very Soluble salt that
does not harm proteins. Refer to the Hofmiester
Series
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Solubility of carboxy-hemoglobin at its
isoelectric point
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Solubility of b-lactoglobulin as a function of pH
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Chromatography

• Analytical methods used to separate molecules.
  Involves a mobile and a stationary phase.
• Mobile phase is what the material to be separated
  is dissolved in.
• Stationary phase is a porous solid matrix which
  the mobile phase surrounds.
• Separation occurs because of the differing
  chemistries each molecule has with both the
  mobile and stationary phase.
• Chemistries are different depending on the
  specific method.

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Types of chromatography

• Gas - Solid Mobile phase is gaseous, stationary
  phase is a solid matrix.
• Liquid - Solid Mobile phase is liquid,
  stationary phase is a solid matrix.
• If separation is based on ionic interaction the
  method is called Ion Exchange chromatography.
• If separation is based on solubility differences
  between the phases the method is called
  adsorption chromatography.
• If the separation is base on size of molecule the
  method is called gel filtration or size
  exclusion.
• If the separation is base on ligand affinity the
  method is called Affinity chromatography.

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Ion Exchange Chromatography

• A solid matrix with a positive charge i.e. R can
  bind different anions with different affinities.
• We can swap one counter ion for another
• (RA-) B- ? (RB-) A-
• R Resin and exchanges Anions (-)
• This is an anion exchange resin.
• There are also cation exchange resins. The type
  of an R group can determine the strength of
  interaction between the matrix, R and the counter
  ion.
• If R is R-
• (R-A) B ? (R-B) A-

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Proteins have a net charge.
The charge is positive below pI, while the charge
is negative above pI The choice of exchange
resin depends on the charge of the protein and
the pH at which you want to do the
purification. Once the protein binds, all unbound
proteins are washed off the column. Bound
proteins are eluted by increasing the ionic
strength, changing the counter ion or changing
the pH altering the charge on the protein or the
column.
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Paper chromatography
Stationary phase vs.. the Mobile
phase Partitioning between the two
phases Partition coefficient The more H2O
soluble the slower it migrates. The more organic
soluble the more it migrates. The aqueous
component of the solvent combines with the
cellulose of the paper and becomes the stationary
phase.
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• Materials can be visualized by
• Radioactivity
• Fluorescence
• UV absorbency
• Stained with one of several dyes
• Ninhydrin
• Iodine
• Sulfuric acid

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Ninhydrin visualizes amino acids
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Two dimensional separation of Amino acids
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Gel FiltrationSize exclusion
A matrix with holes in it. Vt Vx
Vo Vo void volume volume outside the caves
or knooks and crannies Vx occupied by gel
beads Vo ? 35 of Vt
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Gel filtration can be used to determine the
molecular mass of proteins
Ve elution volume Vo exclusion volume Common
matrix dextran, agarose, or polyacrylamide also
desalts proteins
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Before swelling the dry bead size ? 5 of Vt 60
are holes Hole sizes can be made
different Small molecules see a larger column
volume than big molecules and they get hung up in
the caves. Large proteins are excluded, while
small protein are included. Separation on size
and shape.
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Dialysis is a process that separates molecules
according to size through the use of
semipermeable membranes containing pores of less
than macromolecular dimensions
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Affinity Chromatography
Based on molecular complementary between an
enzyme and substrate. The substrate (R) is linked
to a matrix with a spacer arm
Only protein that binds R will stick to column.
put citrate on column citrate dehydrogenase will
specifically bind. Add excess citrate and the
enzyme will be released.
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The purification of Staphylococcal nuclease using
the ligand, diphosphothymadine
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Electrophoresis
The migration of ions in an electric field Fele
qE where q is the charge and E is the electric
Field strength Opposing this is Ffriction vf
where v velocity of migration f is the
frictional force. qE vf
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Paper electrophoresis
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Acrylamide gel electrophoresis
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Disc gel using a glass tube
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Separates on charge and size
pH matters as well as the pI of the protein. Can
be run at several pH values depending on
proteins. DNA can also be separated on agarose
gels. Genomic sized DNA can also be separated but
requires more sophisticated equipment.
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Proteins can be visualized by several methods
Stained with a Dye Coomassie blue Fluorescami
ne stain for fluorescence Silver staining
very sensitive proteins can be labeled with
radioactivity and visualized by exposure
to X- ray film
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SDS-PAGE
Add sodium dodecyl sulfate, a 12 carbon detergent
to give a negative charge to the protein. SDS
also denatures the protein and collapses into a
globular ball. The proteins are separated by
molecular mass
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