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Unit 5

Protein Purification and


Characterization Techniques
Isolation, Purification and Characterization of Proteins

1. Selection of sample
2. Solubilization of protein from sample
3. Purification of protein of interest from homogenized
sample
4. Characterization of protein of interest
Isolation, Purification and Characterization of Proteins

1. Selection of Starting Material


Sources: animal, plant tissues or microorganisms

Criteria for choosing a sample


ease of obtaining sufficient quantity of tissue
amount of biomolecule in the tissue
any properties peculiar to the biomolecule of choice
Isolation, Purification and Characterization of Proteins

2. Solubilization of protein from sample


Homogenization process of rupturing the plasma
membrane (includes bacterial/plant cell wall) to release
the protein from the cell
Isolation, Purification and Characterization of Proteins

3. Purification of protein of
interest from homogenized
sample
Selection of purification
technique to use is based
on proteins unique
structure and chemistry in
order to separate it from
other molecules.
Purification Techniques

Separation based on solubility


1. Isoelectric precipitation
A procedure in which the pH of the protein mixture is
adjusted to the pI of the protein to be isolated to selectively
minimize its solubility.

Solubility of protein to its isolectric point (IEP)


Purification Techniques

Separation based on solubility


2. Salting out
Increase protein solubility
at low ionic strength
(salting in)
Decrease protein solubility
at high ionic strength;
result to competition
between added salt ions
and proteins for molecular
solvation (salting out)
(NH4)SO4 commonly used
for salting out.
Purification Techniques

Separation based on size, weight or density


1. Differential centrifugation
Process of subjecting a
suspension of sample at
greatly increased
gravitational field (centrifugal
force) by rapidly rotating a
receptacle containing the
sample which will lead to
sedimentation of particles.

Different speeds of spin allow


for particle separation
Purification Techniques

Separation based on size, weight or density


2. Dialysis
It is the movement of molecules by diffusion from high
concentration to low concentration.

A process that
separates molecules
by the use of a
semi-permeable
membrane.
Purification Techniques

Separation based on size, weight or density


3. Ultrafiltration
When macromolecular solution is forced under pressure
thru a semi-permeable membrane/disc.

Bacteria retain on the membrane


Purification Techniques
Basis of Column Chromatography
Different compounds distribute themselves to a varying
extent between different phases
Interact/distribute themselves
In different phases
2 phases:
Stationary: samples interacts with this phase
Mobile: Flows over the stationary phase and carries along
with it the sample to be separated
Purification Techniques

Separation based on size, weight or density


4. Gel Filtration (Size Exclusion) Chromatography
Stationary phase composed of cross-linked gel particles.

Extent of cross-linking can be controlled to determine


pore size.

Small molecules enter the beads and are retarded,


while, large molecules cannot enter and so they migrate
faster
Gel Filtration Chromatography
Purification Techniques

Separation based on binding specificity


1. Affinity Chromatography
It is based on the ability of
the protein to interact with
specific molecule (ligand);
uses specific binding
properties of proteins.
Stationary phase has a
polymer that can be
covalently linked to a ligand
that specifically binds to
protein.
Purification Techniques

Separation based on charge


1. Ion Exchange Chromatography
Similar to affinity chromatography

Interaction is based on overall


charge of the molecule

Column is packed with resin with


bound ligand (either positive or
negative in charge)
Ion-Exchange Chromatography
Problem Set 1
1. A mixture of lysine, glycine, alanine, isoleucine and
glutamic acid are separated by ion exchange
chromatography. What is the order of elution of these
amino acids if you use buffer system of pH 7?
a) with a cation exchange resin?
b) with an anion exchange resin?
2. A mixture of proteins: ovalbumin (pI = 4.6), urease (pI =
5.0), and myoglobin (pI = 7.0) was subjected to cation
exchanger using a buffer system of pH of 6.5. What is the
order of elution of these proteins?
Purification Techniques

Separation based on charge


2. Electrophoresis
It is the separation of charged particles in an electric field
(thru a support medium) toward opposite charge.
Support medium could be paper, capillary, or gel (e.g.agarose,
polyacrylamide)
SDS-Polyacrylamide Gel Electrophoresis

SDS: mask the intrinsic charge of


protein due to large negative
charge it imparts on it.
Separates protein in the order of
their MWs
Purification Techniques

Separation based on charge


3. Isoelectric Focusing
It is based on differing isoelectric points of proteins.
Involves electrophoresis of protein mixtures thru stable pH
gradient medium.
Protein will migrate to the region where pH = IpH.
Isolation, Purification and Characterization of Proteins

4. Characterization of protein of interest


Determine the primary structure of the protein
Determine which amino acids are present
(amino acid analysis)

Determine the N- and C- termini of the sequence


(amino acid sequencing)

Determine the sequence of smaller peptide fragments


(most proteins > 100 a.a)
Primary Structure Determination
Amino Acid Analysis
Primary Structure Determination

1. Identification of N-terminal amino acid residue


Sanger Method
Primary Structure Determination

1. Identification of N-terminal amino acid residue


Dansyl chloride
Primary Structure Determination

1. Identification of N-terminal amino acid residue


Edman degradation
Primary Structure Determination

1. Identification of N-terminal amino acid residue


Aminopeptidase

Gly Arg Phe Ile Lys Met Leu

2. Identification of C-terminal amino acid residue


Carboxypeptidase

Gly Arg Phe Ile Lys Met Leu


Primary Structure Determination

3. Cleavage of protein into smaller peptide fragments

Protein cleaved at specific sites by:


Enzymes- Trypsin, Chymotrypsin
Chemical reagents- Cyanogen bromide

Enzymes:
Trypsin- Cleaves @ C-terminal of (+) charged side chains
Chymotrypsin- Cleaves @ C-terminal of aromatics
Peptide Digestion by Trypsin
Peptide Digestion by Chymotrypsin
Cleavage by Cynogen Bromide (CNBr)
Cleaves @ C-terminal of INTERNAL methionines
Determining Protein Sequence
After cleavage, mixture of peptide fragments produced.
Can be separated by HPLC or other chromatographic
techniques
Use different cleavage reagents to help in 1 determination
Problem Set 2
Determine the sequence of a peptide consisting of 14
amino acids on the basis of the following data.

1. Amino acid composition: (4S,2L,F,G,I,M,T,W,Y, K)


2. N-terminal analysis: S
3. Carboxypeptidase digestion: L
4. Trypsin digestion: (3S,2L,F,I,M,T,W) (G,K,S,Y)
5. Chymotrypsin digestion: (F,I,S) (G,K,L) (L,S) (M,T)
(S,W) (S,Y)
6. N-terminal analysis of (F,I,S) peptide: S
7. Cyanogen bromide treatment: (2S,F,G,I,K,L,M*,T,Y)
(2S,L,W)