Transcript CHMI 2227E Biochemistry I

CHMI 2227E
Biochemistry I
Protein purification and
characterization
CHMI 2227 - E.R. Gauthier, Ph.D.
1
Protein purification

Proteins are always found as part of complex mixtures;

While some proteins are quite abundant (antibodies in the blood), others
are found in minute amounts (a few molecules per cell);

The first step to study the structure and/or function of a specific protein
will always be its purification;

From a biological sample containing the protein of interest, a series of
procedures are used sequentially to progressively obtain the protein in
a « pure » form;

Protein purification is made much easier by previous knowledge of:


Basic characteristics of the protein: Mr, pI, solubility;
The availability of an assay to detect the presence of the protein (reagents,
enzymatic reaction, physiological effect, etc).
CHMI 2227 - E.R. Gauthier, Ph.D.
2
Protein purification
General procedure
Crude protein extract
Homogenization
Monitor presence of
protein of interest:
- Activity
- Purity
- Quantity
PURE!
(well, let’s hope so…)
CHMI 2227 - E.R. Gauthier, Ph.D.
Coarse
purification
steps
Chromatography
1, 2, 3….x steps
3
Protein purification
1. Coarse methods

Based on the solubility of the protein of interest under
different conditions:



pH
Temperature
Ionic strength (i.e. salt concentration);

The idea is to use conditions that will lead to the
precipitation of a lot of the proteins of the crude extract,
while keeping the protein of interest in solution;

Allows one to get rid of « most of the junk » in the
extract;
CHMI 2227 - E.R. Gauthier, Ph.D.
4
Differential precipitation
1.1. Precipitation by adjusting the pH
Protein solubility is made possible by
the interactions between the side
chains and the solvent:

If solvent is water:



H bonds
Electrostatic interactions
If solvent is non-polar:

Hydrophobic interactions;

These interactions can be altered by
changing the net charge of the protein,
therefore by adjusting the pH of the
solvent;

As a general rule, proteins
precipitate when the pH of the
solvent equals their pI:

solubility

pI
pH
Proteins aggregate and clump together.
CHMI 2227 - E.R. Gauthier, Ph.D.
5
Differential precipitation
1.2.Salting out

Protein solubility can also be modified
by altering the salt concentration in the
extract;


http://www.tulane.edu/~biochem/med/pure.htm



The added salts will take the place of
the water around the protein;
The salts will also neutralize the
charges on the protein;
Usually, salting out is performed in the
presence of ammonium sulfate:


(NH4)2SO4
More soluble in water than NaCl;
Less amounts are required for protein
precipitation.

Proteins usually precipitate when a
specific concentration of (NH4)2SO4
is reached;

This allows one to clean up the cell
extract in a stepwise fashion;

The precipitated proteins can be
discarded OR sujected to dialysis (to
remove the salt)
These two events will favor protein
aggregation and precipitation;
CHMI 2227 - E.R. Gauthier, Ph.D.
6
Differential precipitation
1.2.Salting out
PRECIPITATION!!
CHMI 2227 - E.R. Gauthier, Ph.D.
7
Differential precipitation
1.2.Salting out
Mixture of proteins in buffer:
A: precipitates at salt [ ] = 15%
B: precipitates at salt [ ] = 25 %
C: precipitates at salt [ ] = 35%
1)
Pellet: protein A
2)

Add (slowly) (NH4)2SO4 to
20%
Centrifuge to pellet
precipitated proteins
Supernatant: protein B + C
1)
Pellet: protein B
2)
Centrifugation:

Separation by way
of density;

Dense (i.e. big)
molecules will
sediment (i.e. pellet)
faster than light (i.e.
small) molecules;
Add (slowly) (NH4)2SO4 to
30%
Centrifuge to pellet
precipitated proteins
Supernatant: protein C
CHMI 2227 - E.R. Gauthier, Ph.D.
8
Centrifugation

Widely used technique which uses centrifugal
force to separate substances;

The centrifugal force applied varies depending on
the substances to be separated:



500 x g for whole cells
15 000 x g for mitochondria
150 000 x g for ribosomes
Note: 1 x g = gravitational force on Earth at sea
level (~ 9.8 m/s2)

Particulate substances will reach the bottom of
the tube (i.e. they form a pellet)

The remaining liquid is called the supernate or
supernatant liquid.

Can also be used to separate substances of
different densities (denser substances will
sediment faster than less dense substances)

e.g. separation of erythrocytes from leukocytes
CHMI 2227 - E.R. Gauthier, Ph.D.
9
Dialysis
Hours

Because the salt used for the
salting out generally inhibits the
activity of proteins, it has to be
removed;

This is done by dialysis;

The protein solution is simply put
is a bag made of a semipermeable membrane:

Permeable to small molecules
(e.g. salts);
 Not permeable to proteins;

Also allows one to change buffer
before performing the next step.
CHMI 2227 - E.R. Gauthier, Ph.D.
10
Protein purification
2. Fine methods

After using crude methods to
concentrate our protein of interest,
other, more refined techniques
must be used to get rid of other
proteins;

Usually achieved by different
types of chromatography:




Buffer reservoir
Column
Sample
injector
Ion exchange;
Molecular sieve;
Affinity
Usually performed by Fast
Protein Liquid Chromatography
(FPLC)
Pumps
CHMI 2227 - E.R. Gauthier, Ph.D.
Fraction collector
11
Protein purification
2.1. Ion exchange chromatography

Allows proteins to be separated
according to their charge;

Cation exchange:




Protein mixture is placed in a buffer,
the pH of which will give a specific, net
charge to the protein of interest;

Phosphocellulose
Carboxymethyl (CM) cellulose
Anion exchange:

Diethylaminoethyl (DEAE) cellulose
The mixture is then loaded onto a
chromatographic column containing
the appropriate separation medium;
CHMI 2227 - E.R. Gauthier, Ph.D.
12
Protein purification
2.1. Ion exchange chromatography
Adsorption
Desorption
NaCl
Anion exchanger
Example of cation
exchange chromatography

Although a change of buffer (with a
different pH) could succeed in eluting the
proteins attached to the column, this is
rarely done since a change in pH could
denature the protein or worse, lead to its
precipitation;

Elution is usually performed by adding a
buffer with a salt (NaCl) concentration that
will prevent protein binding to the column;

Elution can be performed using linear or
stepwise gradients of salt concentration;
CHMI 2227 - E.R. Gauthier, Ph.D.
13
Protein purification
2.1. Ion exchange chromatography
Progressive, linear
change in NaCl
concentration
Add buffer
Stepwise gradient
of NaCl concentration
CHMI 2227 - E.R. Gauthier, Ph.D.
14
Protein purification
2.2. Molecular sieve chromatography

Also called gel filtration chromatography or
size exclusion chromatography;

Uses beads that contain pores of a specific
size:

Proteins larger than the pores cannot enter the
beads, have less volume to go through and
elute first;

Proteins smaller than the pores will enter the
beads and their elution will be delayed: they will
elute later;

Proteins are therefore separated according to
their molecular mass:

The availability of different types of beads
with different ranges of pore size allows one
to select the right chromatographic media for
their separation requirements;
CHMI 2227 - E.R. Gauthier, Ph.D.
15
http://www1.amershambiosciences.com/aptrix/upp00919.nsf/Content/LabSep_Prod~SelGuides~Media~GFSelG
CHMI 2227 - E.R. Gauthier, Ph.D.
16
Protein purification
2.2. Molecular sieve chromatography

Gel filtration has many applications:

1) Protein purification

2) Desalting



NOT the same thing as salting out;
The pores are so small than all the
proteins go through and only the
elution of salts is delayed;
3) Molecular mass determination:


Requires one to run a set of
standards first (a mixture of proteins
of known Mr);
Allows one to determine whether
his/her favorite protein is part of a
multimeric complex. WHY?
CHMI 2227 - E.R. Gauthier, Ph.D.
17
Protein purification
2.3. Affinity chromatography

In this type of chromatography, beads are linked
to a molecule called a ligand, which can only
bind the protein of interest;

Ligand: antibody, substrate, metals, other
protein/macromolecule interacting with the
protein of interest.

When a mixture of partially purified proteins is
filtered through this type of column, only the
protein of interest is bound. The other,
contaminating proteins are washed out;

The protein of interest is then eluted by the
addition of an excess of unbound ligand;

Very powerful method but:



Media is expensive (cannot perform large scale
purifications)
Limited availability of ligand-bonded beads;
Requires prior knowledge of a ligand that can bind
the protein of interest (which is seldom the case).
CHMI 2227 - E.R. Gauthier, Ph.D.
18
Analysis of proteins
Electrophoresis

During the purification process, the presence and purity of the protein of
interest needs to be assessed;

Also, methods are required to study proteins without the need to purify
them;

The most popular and simple methods for analyzing rely on the principle of
electrophoresis;

Electrophoresis involves the separation of proteins via their migration
through a gel, under an electric field.

The material generally used to separate proteins by electrophoresis is
polyacrylamide.
CHMI 2227 - E.R. Gauthier, Ph.D.
19
Electrophoresis
1. SDS-PAGE Electrophoresis

In SDS-PAGE, proteins are first placed in a buffer
containing the detergent sodium dodecyl sulphate
(SDS);

SDS will cover each protein similarly: approx 1 SDS
molecule for every 2 amino acids;

This has the consequence of giving each protein the
same density of negative charges, independent of
their pI.

b-mercaptoethanol (HS-CH2-CH2-OH) is also
frequently (but NOT always) added to break disulfide
bonds.
SDS

Therefore, the only variable that can modify the
migration of proteins in a gel will be their size;
CHMI 2227 - E.R. Gauthier, Ph.D.
20
Electrophoresis
1. SDS-PAGE Electrophoresis

The proteins are then placed in the wells of a SDS-PAGE gel, and subjected to an
electric field;

The proteins then migrate towards the positively-charged anode: the distance they
will migrate depends on their size;

Proteins are then stained in the gel, generally with a blue dye called Coomassie
Blue;

By placing in an adjacent well a mixture of proteins of known molecular mass (protein
standards), one can determine the molecular mass of the protein of interest.

Of note: treatment with SDS and b-ME denatures the proteins and breaks all
interactions with other proteins: the protein will migrate as if it were a single
polypeptide.

Sometimes, NATIVE PAGE is performed: in this situation, no SDS or b-ME is added.
This allows proteins part of multimeric complexes to be studied. WHY?
CHMI 2227 - E.R. Gauthier, Ph.D.
21
Electrophoresis
1. SDS-PAGE Electrophoresis
www.biology.ucsd.edu/classes/bggn224.FA06/crash_2.ppt
http://wine1.sb.fsu.edu/bch5425/lect20/lect20.htm
CHMI 2227 - E.R. Gauthier, Ph.D.
22
Electrophoresis
1. SDS-PAGE Electrophoresis
CHMI 2227 - E.R. Gauthier, Ph.D.
23
Electrophoresis
Log Mr
1. SDS-PAGE Electrophoresis
Distance migrated from well (cm)
CHMI 2227 - E.R. Gauthier, Ph.D.
24
Electrophoresis
2. Isoelectrofocusing

Here, proteins are separated according to their pI;

The mixture of proteins is placed in the wells of an acrylamide gel
containing a gradient of pH along its length;

Upon subjecting the proteins to an electric field, they will migrate in the gel
according to their charge:


Positively charged proteins migrate towards the negative cathode;
Negatively charged proteins migrate towards the positive anode;

When a protein reaches a zone in the gel where the pH = pI, it carries no
net charges and stops migrating;

This allows one to determine the pI of the protein of interest;
CHMI 2227 - E.R. Gauthier, Ph.D.
25
Electrophoresis
2. Isoelectrofocusing
pH in gel = pI of proteins
CHMI 2227 - E.R. Gauthier, Ph.D.
26
Electrophoresis
3. Two-dimensional gel electrophoresis

Here, the proteins are FIRST
separated according to their pI
by IEF.

THEN, the strip of IEF gel is
placed on top of a PAGE-SDS
gel: the proteins will then be
separated according to their
Mr;

Allows a much higher
resolution of the proteins
present in the mixture of
interest;
CHMI 2227 - E.R. Gauthier, Ph.D.
27
Electrophoresis
3. Two-dimensional gel electrophoresis
Each spot is a
single protein
CHMI 2227 - E.R. Gauthier, Ph.D.
28
Electrophoresis
4. Western blot analysis

This is a powerful application of SDSPAGE;

Allows the detection of a single protein
of interest present in a complex
mixture of proteins;

Makes use of the properties of
antibodies to specifically bind a
unique molecule with high affinity;

Antibodies:

4 chains: 2 light chains (25 kDa ea)
and 2 heavy chains (50 kDa each);

Each antibody possesses two identical
binding site for an antigen (antigen:
whatever the antibody specifically and
uniquely binds to)
Structure of an antibody
CHMI 2227 - E.R. Gauthier, Ph.D.
29
Electrophoresis
4. Western blot analysis
CHMI 2227 - E.R. Gauthier, Ph.D.
30
Protein purification
Example and data analysis

At each step, took 3
samples:




One for protein
quantification
One for protein activity
(assay)
One for PAGE-SDS
The rest was subjected to
the next purification step;
CHMI 2227 - E.R. Gauthier, Ph.D.
31
Protein purification
Example and data analysis
Coomassie-blue-stained
PAGE-SDS gel
CHMI 2227 - E.R. Gauthier, Ph.D.
32
Protein purification
Example and data analysis

Important values to obtain to get an estimation of the success of the
purification (especially when setting up the procedure):

Specific activity (units/mg): Total activity (U)/ Total protein (mg)
 Yield: (Total activity at Step Y / Total activity in crude extract) x 100;
 Purification level: Specific activity at Step Y / Specific activity in crude
extract;
CHMI 2227 - E.R. Gauthier, Ph.D.
33
Protein sequencing

Every functional and structural properties depend on the order of amino
acids in the polypeptides (their sequence);

The 3-D structure of a protein is uniquely dictated by its amino acid
sequence;

Several genetically-heritable diseases are caused by a change (insertion,
deletion, substitution) of one or more amino acids in the sequence of one
protein (e.g. DY508 in cystic fibrosis);

Amino acids important in the structure/function of a protein will not change
rapidly during evolution.

Comparisions between the amino acid sequences of proteins from different
species can reveal unknown functions/properties of the protein of interest.
CHMI 2227 - E.R. Gauthier, Ph.D.
34
Protein sequence
- Sickle-cell anemia

Hemoglobin is a tetramer made of 2 copies each
of 2 polypeptides: HbA and HbB

Sickle cell anemia is caused by a single, heritable
mutation in HbB:

Normal red blood cell
Glu replaced by Val

Creates a hydrophobic patch (why?) that causes
the aggregation of the mutated hemoglobin (called
HbS).

This aggregated (i,e, clumped) HbS forms long
fibers that change the shape of the erythrocytes.
These elongated red blood cells hinder blood flow.
These elongated blood cells are also very fragile
and burst easily (hence the anemia).

BUT: because the malaria parasite grows in
erythrocytes, people with sickle cell anemia are
more resistant to malaria.
CHMI 2227 - E.R. Gauthier, Ph.D.
Sickled red blood cell
35
Determination of protein sequence
1. Enzyme mapping
Enzyme
Trypsin
Chymotrypsin
Protease V8
Pepsin
Thermolysin
Carboxypeptidase
A
Carboxypeptidase
B
Amino acid
Cutting
site
Arg/Lys
C-ter
Phe/Trp/Tyr
C-ter
Asp/Glu
C-ter
Phe/Trp/Tyr
N-ter
Leu/Ile/Trp/Tyr/
Val/Ala/Phe
N-ter
All C-ter a.a.
except Pro,
Arg/Lys
Only Arg/Lys
when C-ter
- Free amino
acids from
the C-ter
- Doesn’t cut
if Pro is the
penultimate
amino acid
NOTE: Trypsin, Chymotrypsin, protease V8,
pepsin and thermolysin do NOT cut if Pro is part of
the peptide bond.

Based on the property of
some enzymes to cut the
peptide bonds next to
specific amino acids;
Chemical
Amino
acid
Cutting site
Cyanogen bromide
Met
C-ter
b-mercaptoethanol
Cys
Disulfide bonds
Iodoacetate
Cys
Prevents the
reduction of
disulfide bonds
1) 1-Fluoro-2,4
dinitrobenzene (FDNB)
2) Dansyl chloride
3) Dabsyl chloride
Destroy all the amino acids
with the exception of the one
at the N-terminus.
Hydrazine
Destroy all the amino acids
with the exception of the one
at the C-terminus.
36
CHMI 2227 - E.R. Gauthier, Ph.D.
Determination of protein sequence
1. Enzyme mapping – example 1
CHMI 2227 - E.R. Gauthier, Ph.D.
37
Determination of protein sequence
1. Enzyme mapping – example 2


The following data were obtained after treating an octopeptide with
the following reagents:
HCl 6M: Ala, Gly2, Lys, Met, Ser,
Thr, Tyr

Chymotrypsin: 2 peptides were
obtained:





Peptide 1: Ala, Gly, Lys, Thr
Peptide 2: Gly, Met, Ser, Tyr
Trypsin: 2 peptides were obtained:




CNBr: 2 peptides were obtained:
Peptide 3: Ala, Gly
Peptide 4: Gly, Lys, Met, Ser, Thr,
Tyr
Peptide 5: Gly, Tyr
Peptide 6: Ala, Gly, Lys, Met, Ser,
Thr

FDNB: yields Gly

Carboxypeptidase A: yields Gly
What is the sequence of this peptide?
CHMI 2227 - E.R. Gauthier, Ph.D.
38
Determination of protein sequence
2. Edman degradation

Based on the use of phenyl
isothiocyanate (aka PTC; Edman’s
reagent);

PTC reacts with and labels the amino
acid at the N-terminus of the peptide;

The PTC-labeled amino acid is released
from the peptide and identified by
chromatography;

Cycles of labeling and release allow one
to determine the sequence of the
peptide.
CHMI 2227 - E.R. Gauthier, Ph.D.
39
Determination of protein sequence
2. Edman degradation
Identify
CHMI 2227 - E.R. Gauthier, Ph.D.
40
Determination of protein sequence
3. Mass spectrometry

Very powerful, now trendy technique to identify and sequence proteins;

Proteins are vaporised into ionized fragments with the use of a laser;

Even proteins cut out of a SDS-PAGE gel can be use!!!

The fragments are separated and their molecular mass determined;

From the molecular mass of the fragments, the peptide can be identified;

In tandem MS (MS-MS), fragments obtained after one MS run are subjected
to a second fragmentation into even smaller fragments: the mass of these
smaller fragments can be used to determine the amino acid sequence of the
fragment.
CHMI 2227 - E.R. Gauthier, Ph.D.
41
Determination of protein sequence
3. Mass spectrometry
CHMI 2227 - E.R. Gauthier, Ph.D.
42
Determination of protein sequence
3. Mass spectrometry
CHMI 2227 - E.R. Gauthier, Ph.D.
43
Example of protein purification
Apoptosis: a form of cell death
Current Opinion in Cell Biology 2004, 16:663–669
Normal
Apoptosis
CHMI 2227 - E.R. Gauthier, Ph.D.
Autophagy
Necrosis
44
http://www.nature.com/nrm/journal/v9/n3/extref/nrm2312-s1.mov
Apoptosis – morphological aspects
45
Shrinkage
Blebbing
Fragmentation
Apoptosis – morphological aspects
http://www.nature.com/nrm/journal/v9/n3/extref/nrm2312-s1.mov
46
Apoptosis and physiology
http://www.wikiwak.com/image/Celldeath.jpg
47
Example of protein purification
Acinus: a protein involved in cell death
NATURE |VOL 401 | 9 SEPTEMBER 1999
CHMI 2227 - E.R. Gauthier, Ph.D.
48
Chromatin condensation and nuclear
fragmentation during apoptosis
Nature Reviews| molecular cell biology volume9 | march2008 | 231
49
Example of protein purification
Acinus: a protein involved in cell death
CHMI 2227 - E.R. Gauthier, Ph.D.
50
Example of protein purification
Acinus: a protein involved in cell death






Lane 1, 100,000g supernatant
(3.4mg);
lane 2, HiTrap-Q(1.7mg);
lane 3, Heparin Sepharose after
passing the hydroxyapatite
column (150 ng);
lane 4, Phenyl Sepharose (70
ng);
lane 5, Superose 12 (50 ng);
lane 6, Mono-Q (2.5 ng).
Arrowhead, position of purifed
Acinus p17 protein.
CHMI 2227 - E.R. Gauthier, Ph.D.
51
Example of protein purification
Acinus: a protein involved in cell death
CHMI 2227 - E.R. Gauthier, Ph.D.
52
Example of protein purification
Acinus: a protein involved in cell death
CHMI 2227 - E.R. Gauthier, Ph.D.
53