PROTEIN PURIFICATION

M.Prasad Naidu
MSc Medical Biochemistry, Ph.D,.

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.
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 | (1-4) linkages in
the (glycosidic bonds) of polysaccharides.
Mechanical breakage blenders homogenizers
French press - high pressure 20,000 lbs/in
2
forced
through a small hole disrupts cells
ultrasound or sonication disrupts cells.
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




Centrifugation: Units




( )
Nf
V M
dt
r d
r
v
s

e e

=
|
.
|

\
|
= =
1 ln 1
2 2
Where:
e = angular velocity
v = velocity of particle
R = distance from center of rotation
M = molecular weight
V = partial specific volume of particle
= density of solvent
Sedimentation velocity (Svedberg
Coefficient)
S = s x 10
-13

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 0
2
Heavy and transition metals damage proteins -they bind to
protein- Cu
+
Hg
+

Bacterial contamination can destroy the protein
STABILITY: PROTEINS CAN
DENATURE!!
In order to follow the purity of an enzyme, you
need a method to measure its activity.

Spectraphotometric 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 = cb c
c - concentration
c - millimolar extinction coefficient
A - absorbance
b - path length
T - percent transmittance
ACTIVITY MEASUREMENTS
A = - log % T
if A A then A c at max
For the reaction: NADH NAD
+
+ H
-

enzyme
A
b
s
o
r
b
a
n
c
e

300 nm 350 nm
NADH
NAD
+

AA
Max = 340 nm
oxidized NADH millimolar
A
=
A
c
AT min
mg
mg of protein
}
= Specific activity
Volume is 1 ml so micromoles
NADH oxidized
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 A = 0.5 A/min

1 millimolar = 6.22 AA = c 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

AC=.008 moles in 1 ml/min = .04 moles
0.2 mg min/mg

Total activity: .04 moles x 20 mg/ml = 0.8 moles / 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.
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:

enz
1
enz
2

A B C

NADH NAD
+


Coupled Reactions: We couple enz
2
to enz
1
and measure ANADH to get
AA
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
N
N
N
N
NH
2
O
H OH
H H
H H
O P -O
O-
O
O P -O
O-
O
O- O P -O
O-
O
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

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

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

2
i
Z c
2
1
I
i

=
For di- or tri-valent ions, where I is different than M


1M MgCl
2

Mg
++
= 1M, and Z = 2
while Cl
-
= 2M, and Z =1

I = (1 x 2
2
)
Mg
+ (2 x 1
2
)
Cl
= 4 + 2 = 3
2 2

SALTING OUT
Use (NH
4
)
2
SO
4
: it is a Very Soluble salt that does not
harm proteins.
Refer to the Hofmiester Series
SOLUBILITY OF CARBOXY-HEMOGLOBIN AT
ITS ISOELECTRIC POINT
SOLUBILITY OF B-LACTOGLOBULIN AS A
FUNCTION OF PH
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.
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.
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

(R
+
A
-
) + B
-
(R
+
B
-
) + 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
-

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.
PAPER CHROMATOGRAPHY
Stationary phase vs.. the Mobile phase
Partitioning between the two phases




Partition coefficient
The more H
2
O 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.
| |
| | phase mobile in A
phase stationary in A
=
p
K
front solvent by the traveled distance
substance by the traveled distance
=
f
R
Materials can be visualized by:
Radioactivity
Fluorescence
UV absorbency
Stained with one of several dyes
Ninhydrin
Iodine
Sulfuric acid
NINHYDRIN VISUALIZES AMINO ACIDS
TWO DIMENSIONAL SEPARATION OF
AMINO ACIDS
GEL FILTRATION
SIZE 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
Ve = elution volume Vo = exclusion volume
Common matrix: dextran, agarose, or polyacrylamide
also desalts proteins
Gel filtration can be used to determine
the molecular mass of proteins
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.
Dialysis is a process that separates molecules according to size
through the use of semipermeable membranes containing
pores of less than macromolecular dimensions
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.
The purification of Staphylococcal nuclease using
the ligand, diphosphothymadine
Electrophoresis
The migration of ions in an electric field
F
ele
= qE where q is the charge
and E is the electric Field strength
Opposing this is F
friction
= vf where v =
velocity of migration f is the frictional
force.
qE = vf
f
v q
E
= =
PAPER ELECTROPHORESIS
ACRYLAMIDE GEL ELECTROPHORESIS
DISC GEL USING A GLASS TUBE
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.
PROTEINS CAN BE VISUALIZED BY SEVERAL
METHODS
Stained with a Dye: Coomassie blue
Fluorescamine stain for
fluorescence
Silver staining very sensitive
proteins can be labeled with
radioactivity
and visualized by exposure to X-
ray film
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