Protein Purification Strategies
Course: Methods in protein chemistry
Rahman M. Mahfuzur
2012/01/11
SLU
Objectives
To seperate a particular protin from all other proteins
and cell components
There are many types of proteins within an ogranism
Other components: nuclic acids, charbohydrates,
lopids, small molecules
A gieven protein could be 0.001-20% of total protein.
Protein purification
Proteins purification varies from one purification step to
multi- step of purifixcations
Often more than one purification step is necessary to
reach the desired purity, or step can be repetead if
sample is available.
Successful and efficient protein purification depends on
appropriate methods selection.
Methods should be sequence in a logical manner,
what kinds of materials are available/handle?
what has to be removed/ completely?
what will be the use of final products?
what are economical constraints?
Three phase purification strategies(CIPP)
The four parameters
Every technique offers a
balance between resolution,
capacity, speed and recovery
Capture
Initial purification of target
Rapid isolation, stabilization,
concentration
Intermediate purification
Further removal of bulk
contaminants: other proteins,
nucleic acids, endotoxins
and viruses.
Purification and concentration.
Polishing
Final removal of remaining trace
impurities or closely related
substances
to achieve high purity
Protein properties Vs Technique
Protein property
Technique
Charge
Ion exchange (IEX)
Specific ligand recognition
(biospecifc or nonbiospecifc)
Affinity chromatography (AC)
Size
Gel filtration (GF)
Hydrophobicity
Hydrophobic interaction (HIC),
Reversed phase (RPC)
Isoelectric point
Chromatofocusing
Ion exchange chromatography (IEX)
separates proteins with
differences in surface charge.
based on the reversible
interaction, charged protein Vs
oppositely charged column
If, pHb>PIP Neg. bind 
positively charged anion
exchanger ; ex- MonoQ column
When, pHb<PIP Pos. bind 
negatively charged cation
exchanger ; ex- MonoS column
IEX chromatogram
Affinity chromatography (AC)
On the basis of a reversible
or specific interaction
between target and a
specifc ligand
Biospecific: antibodies
binding proteins,
Non-biospecific: histidine
binding protein bind to
metal Ion; IMAC
AC chromatogram
Gel filtration chromatography (GF)
allow separation of
proteins with differences in
molecular size
GF is a non-binding method
0.5% to 2% of total column
volume.
FG chromatogram
Hydrophobic interaction
chromatography (HIC)
separates proteins with
differences in hydrophobicity
based on the reversible
interaction between a
protein and the hydrophobic
surface of a chromatography
medium
Technique Vs three phase
combinations of chromatographic
steps
IEX-HIC-GF
Considered as a standard
protocol
If nothing is known about
the target protein use IEXHIC-GF.
both anion and cation
exchange could be used to
get different selectivities
within the strategy.
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