Chapter Five

Protein Purification and

Characterization Techniques
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 (Cont’d)
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 (Cont’d)

• 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 electric-
field. 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