Transcript How to assemble a protein sequence

Protein Purification
February 5 2003
“A basic comprehension of the
methods described here is
necessary for an appreciation of
the significance and the
limitations of the information
presented in the text”
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. baker’s 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 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.
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

1  d ln r  M 1  V 
s 2  2

 r   dt 
Nf
v
Where:
 = 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

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
Activity Measurements
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 Beer’s Law.
A = eb c
A = - log % T
c - concentration
e - millimolar extinction coefficient
A - absorbance
b - path length
if  A then  c at  max
T - percent transmittance
enzyme
For the reaction: NADH  NAD+ + HAbsorbance
NADH
A
NAD+
300 nm
A
e
T
mg
 Max = 340 nm
 millimolar
350 nm
Volume is 1 ml so micromoles
NADH oxidized
NADH oxidized
min
mg of protein
}
= Specific activity
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 A = 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
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:
A
enz1
B
NADH
enz2
C
NAD+
Coupled Reactions: We couple enz2 to enz1 and measure NADH to get
A
Use of radioactivity
NH 2
ATP  ADP + Pi
O
-O
P
O-
O
O
O-O
P
O-
N
N
O
O
-O
P
O
N
N
O
O-
H
H
OH
H
H
H
Separate ATP + Pi + ADP on TLC measure radioactivity
Pi
ATP
Phosphoimager makes this easy else cut spots and count in
scintillation counter.
Strategy of Purification
Fractionation procedures or steps to isolate protein based on
physical characteristics.
Characteristic
•Charge
Procedure
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
1
2
I   c i Zi
2
Ci = the molar concentration of the ith species
Zi = it’s ionic charge
1M Na+ Cl-
Z = 1 Na+
Z = 1 Cl-
1 = (1M x 1)Na + (1M x 1)Cl
2
For di- or tri-valent ions, where I is different than M
1M MgCl2
while
Mg++ = 1M, and Z = 2
Cl- = 2M, and Z =1
I = (1 x 22)Mg + (2 x 12)Cl = 4 + 2 = 3
2
2
Salting out
Use (NH4)2 SO4 : 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-) + AR = 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

A in stationary phase
Kp 
A in mobile phase
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.
distance traveled by the substance
Rf 
distance traveled by the solvent front
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
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
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
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.
v q
 
qE = vf
E f
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 Xray 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