Mary K.

Campbell
Shawn O. Farrell
http://academic.cengage.com/chemistry/campbell

Chapter Five
Protein Purification and
Characterization Techniques

Paul D. Adams University of Arkansas

Isolation of Proteins from Cells

Many different proteins exists within one cell

Many steps needed to extract protein of interest, and

separate from many contaminants
Before purification begins, protein must be released
from cell by homogenization

How We Get Proteins Out of Cells

Salting Out
After Proteins solubilized, they can be purified based
on solubility (usually dependent on overall charge,
ionic strength, polarity
Ammonium sulfate (NH4SO4) commonly used to salt
out

Takes away water by interacting with it, makes protein

less soluble because hydrophobic interactions among
proteins increases
Different aliquots taken as function of salt
concentration to get closer to desired protein sample
of interest (30, 40, 50, 75% increments)
One fraction has protein of interest

Differential Centrifugation
Sample is spun, after
lysis, to separate
unbroken cells, nuclei,
other organelles and
particles not soluble in
buffer used
Different speeds of
spin allow for particle
separation

Column Chromatography
Basis of 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

Column Chromatography

Size-Exclusion/Gel-Filtration
Separates molecules based on size.
Stationary phase composed of cross-linked gel
particles.
Extent of cross-linking can be controlled to determine
pore size
Smaller molecules enter the pores and are delayed in
elution time. Larger molecules do not enter and elute
from column before smaller ones.

Size Exclusion/Gel-filtration (Contd)

Affinity Chromatography
Uses specific binding properties of molecules/proteins
Stationary phase has a polymer that can be covalently
linked to a compound called a ligand that specifically
binds to protein

Ion Exchange
Interaction based on overall charge
(less specific than affinity)
Cation exchange
Anion exchange

Electrophoresis
Electrophoresis- charged particles migrate in electric
field toward opposite charge

Proteins have different mobility:

Charge
Size
Shape

Agarose used as matrix for nucleic acids

Polyacrylamide used mostly for proteins

Electrophoresis (Contd)
Polyacrylamide has more resistance towards larger
molecules than smaller
Protein is treated with detergent (SDS) sodium
dodecyl sulfate
Smaller proteins move through faster (charge and
shape usually similar)

Isoelectric Focusing
Isolectric focusing- based on differing isoelectric pts.
(pI) of proteins

Gel is prepared with pH gradient that parallels electricfield. What does this do?
Charge on the protein changes as it migrates.
When it gets to pI, has no charge and stops

Primary Structure Determination

How is 1 structure determined?
1) Determine which amino acids are present (amino
acid analysis)
2) Determine the N- and C- termini of the sequence
(a.a sequencing)
3) Determine the sequence of smaller peptide
fragments (most proteins > 100 a.a)

4) Some type of cleavage into smaller units necessary

Primary Structure Determination

Protein Cleavage
Protein cleaved at specific sites by:
1) Enzymes- Trypsin, Chymotrypsin
2) Chemical reagents- Cyanogen bromide

Enzymes:
Trypsin- Cleaves @ C-terminal of (+) charged side
chains
Chymotrypsin- Cleaves @ C-terminal of aromatics

Peptide Digestion

Cleavage by 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

Peptide Sequencing
Can be accomplished by Edman Degradation
Relatively short sequences (30-40 amino acids) can
be determined quickly
So efficient, today N-/C-terminal residues usually not
done by enzymatic/chemical cleavage

Peptide Sequencing