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• 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
N-terminal sequencing,
antigen for antibody Moderately high < 95%
production, NMR
Separation of proteins based on
physical and chemical properties
• Solubility
• Binding interactions
• Isoelectric Point
Cell growth,
protein over-
expression
Cell lysis
Removal of
cell debris
Expression of Target Protein in E. coli
DinI
transformation expression
E. coli
Protein isolation, concentration, and
stabilization
Reversible
precipitation
with salt or
organic
molecules
Fractional precipitation of proteins
Discard pellet
Precipitate
contaminants
Precipitate Discard
protein of supernatant,
interest Resuspend
protein
Intermediate Purification
Liquid
chromatography
(lower resolution,
lower cost)
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
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 #
Polishing steps
Liquid
chromatography
(higher resolution,
higher cost)
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
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
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
Final steps in purification
• Check purity by detection methods
• Test for interfering contaminants
– Nucleases
– Proteases
– Toxins
Liquid
chromatography
(higher resolution,
higher cost)
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
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
Ion-exchange Chromatography
• Cation exchangers
contain negatively
charged polymer
• Anion exchangers
contain positively
charged polymer.
• Is effected by pH
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
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
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
SDS-PAGE: Sodium Dodecyl Sulfate (SDS)
Polyacrylamide Gel Electrophoresis
• Separates proteins
of identical MW
that differ in pI or
proteins with
similar pI but
different MW.
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
Bacterial expression vectors
Bacterial expression vectors
Mammalian expression vector