2. Levels of Structure in Protein
• Primary: A description of all covalent bonds. The
sequence of AA residues
• Secondary: particularly stable arrangements of AA
giving rise to recurring structural patterns.
• Tertiary: All aspects of the 3D folding of a polypeptide.
• Quaternary: The spatial arrangement of multisubunits
protein
3. Protein Purification
• Crude extract: breaking cells, by osmosis
lysis or homogenization.
• Fractionation: separate proteins into
different fraction based on size of charge.
• Salting out: The solubility of proteins is
lowered at high salt concentration.
Ammonium sulfate ((NH4)2SO4).
• Dialysis is a procedure to separate
proteins from solvents
4. Guidelines for protein purification
• Define objectives
• Define properties of target protein and
critical contaminants
• Minimize the number of steps
• Use a different technique at each step
• Develop analytical assays
Adapted from: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition AC
5. How pure should my protein be?
Application Required Purity
Therapeutic use, in vivo
Extremely high > 99%
studies
Biochemical assays, X-ray
High 95-99%
crystallography
N-terminal sequencing,
antigen for antibody Moderately high < 95%
production, NMR
6. Separation of proteins based on
physical and chemical properties
• Solubility
• Binding interactions
• Surface-exposed hydrophobic residues
• Charged surface residues
• Isoelectric Point
• Size and shape
7. Basic scheme of protein purification
From: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition AC
13. Types of liquid chromatography
Adsorption Chromatography
– Proteins bind to stationary phase
– Proteins eluted by altering mobile phase
– Includes: affinity, hydrophobic interaction, ion exchange, and
chromatofocusing
Solution Phase Chromatography
– Proteins do not bind to stationary phase
– Progress of proteins through column impeded by matrix of
stationary phase
– Includes: size exclusion chromatography (aka gel filtration)
14. Types of liquid chromatography
Adsorption Resin Chemical Equilibrate Elution
Type Separation By Names of Resins
or Solution Group With With
Metal, Ig,
Low High Hydroxyapatite,
Affinity Adsorption Specific Ligand Ligand Binding
[Ligand] [Ligand] Heparin Sepharose,
Any ligand
Butyl sepharose, Octyl
Hydrophobic Hydrophobic Hydrophobic
Adsorption High Salt Low Salt sepharose, Phenyl
Interaction Groups Effect sepharose
Positively charged Coulombic Mono-Q, Source-Q,
Anion Exchange Adsorption Low Salt High Salt
ions Interacions DEAE
Negatively charged Coulombic Mono-S, Source-S,
Cation Exchange Adsorption Low Salt High Salt
ions Interacions CM
Negatively charged pH
Chromatofocusing Adsorption Isoelectric Point Poly-buffer Mono-P
ions gradient
Size Exclusion Solution Size / Shape of Sephacryl #,
Pores Same Buffer
(gel filtration) Phase Protein Sephadex #
15. Polishing steps
Liquid
chromatography
(higher resolution,
higher cost)
From: Protein Purification Handbook. Amersham Biosciences. 18-1132-29, Edition AC
16. Liquid chromatography techniques
advantages and disadvantages
Type of
Advantages Disadvantages Resolution
Chromatography
Resins and ligands
Affinity Quick and specific
can be expensive Low to Medium
Can be used directly Relatively low
Hydrophobic
Interaction
from ammonium resolution and binding Low to Medium
sulfate precipitation capacity
Protein solution must
Ion Exchange Versatile resin choices
start at low [salt] Medium to High
pH gradient can be
Chromatofocusing High resolution
harsh for protein High
Distinct from other
techniques, Can be
Size Exclusion used analytically or for
Long run time Low to High
buffer exchange
17. Protein detection methods
• SDS-PAGE
– Visual confirmation
• UV Spectrophotometry
– Absorbance @ 280 nm
– Due mostly to Trp
– [Protein] calculated with Beer’s Law
• Colorimetric Techniques
– Color change proportional to [protein]
– Bradford, Lowry, BCA
J.S.C. Olson and John Markwell. Current Protocols in Protein Science (2007) 3.4.1-3.4.29
18. Final steps in purification
• Check purity by detection methods
• Test for interfering contaminants
– Nucleases
– Proteases
– Toxins
• Concentrate your protein
– Precipitation
– Centricons
– Small column with high binding capacity
• Choose a storage buffer and storage conditions
– Consider intended use of protein
– Stabilizing additives
– Flash freeze protein and store at -80o C
• Confirm identity of purified protein
– Mass spectrometry
– N-terminal sequencing
– Analytical assays
19. Basic scheme of protein purification
Cell growth, Liquid
protein over- chromatography
expression Reversible (lower resolution,
precipitation lower cost)
Cell lysis with salt or
Removal of organic
cell debris molecules
Liquid
chromatography
(higher resolution,
higher cost)
20. Separation Processes that can be Used
to Fractionate Proteins
Separation Process Basis of Separation
Precipitation ammonium sulfate solubility
polyethyleneimine (PEI) charge, size
isoelectric solubility, pI
Chromatography gel filtration (SEC) size, shape
ion exchange (IEX) charge, charge distribution
hydrophobic interaction(HIC) hydrophobicity
DNA affinity DNA binding site
immunoaffinity (IAC) specific epitope
chromatofocusing pI
Electrophoresis gel electrophoresis (PAGE) charge, size, shape
isoelectric focusing (IEF) pI
Centrifugation sucrose gradient size shape, density
Ultrafiltration ultrafiltration (UF) size, shape
21. Protein Purification: Column Chromatography
• The expansion of the
protein band in the mobile
phase is caused by
separation of proteins with
different properties and by
diffusional spreading. As
the length of the column
increases, the resolution
of two types of protein
improves.
• Rate is decreased and
resolution can decline
because of the diffusional
spreading
23. Size-Exclusion Chromatography
• Also called gel
filtration: The column
matrix is a cross-
linked polymer with
pores of selected size.
• Larger protein migrate
faster than smaller
ones because they
are too large to enter
the pores
24. Affinity Chromatography
• Separate protein by
their binding
specificities. The
proteins retained on
the column are those
that bind specifically
to a ligand cross-
linked to the beads.
Proteins that do not
binds to ligands are
washed through to
column
25. Electrophoresis
• Separation of porteins is
based on the migration of
charged protein in an electric
field
• The migration of a protein in a
gel during electrophoresis is a
function of its size and shape
µ = V/E = Z/ f
µ : electrophoretic mobility
V: velocity; E: electrical potential
Z: net charge; f: frictional
coefficient
26. SDS-PAGE: Sodium Dodecyl Sulfate (SDS)
Polyacrylamide Gel Electrophoresis
• SDS binds to most proteins probably by
hydrophobic interaction. One SDS for every two
AAs, Thus, each protein has a similar charge-
to-mass ratio.
• Coomassie blue stains protein. Western blot
28. Isoelectric Focusing
• pI of a protein: net
charge=0
• A pH gradient is
established by
allowing a mixture of
organic acids and
bases (ampholytes).
Protein migrates until
it reaches the pH that
matches its pI
29. Two-Dimensional Electrophoresis
• Separates proteins
of identical MW
that differ in pI or
proteins with
similar pI but
different MW.
30. Activity Vs. Specific Activity
• Unit: amount of
enzyme causing
transformation of 1 µ
mole of substrate per
min. at 25 oC under
optimal conditions
• Activity: Total units of
enzyme (U).
• Specific activity:
(U/mg) of total protein
