Download Protein Separation and Purification

Protein Separation and Purification
Methods rely on specific properties of protein
Why purify a protein? Isolate
Allows:
Analysis of the biological properties
Understand its structure
Study interactions
No single procedure can be used to isolate every protein
Exploit specific characteristics (structure or function) of the
protein. Different steps should exploit a different characteristic
Ensure method has little/no effect on function
Early steps involve releasing your protein from the cells
(normally by homogenisation) and low resolving procedures to
remove the bulk of the unwanted proteins
Lysozyme
Sonicator
French Press
Removing crude extract
Ammonium sulphate precipitation (40%)
exploits changes in the solubility of proteins as consequence of a
change in ionic strength (salt conc.) of the solution
At low salt, the solubility of a protein increases with salt
concentration, ‘SALTING IN’.
But as salt conc. (ionic strength) is increased further, the solubility
of the protein begins to decrease, until a point where the protein is
precipitated from solution, ‘SALTING OUT’.
Low salt
Strong attractive force
+
+ +
+ +
+
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+
Optimal salt conc. for solubility
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+
Weak attractive force
+ +
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+ +
- - +
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High Salt
Compete for water
++ + +
-- -- + ++
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++
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-+ +- - -- - +
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+
- -+-+ + +
+
+
- + -++
-++- +
+
- - -++
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-++
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-+-+-+ ++
++
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+ +- + +++ - -+- +
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- - +-+- - -++- - - -- - ++++ ++++ + -- - Debye-Huckel Theory
Ammonium sulphate precipitation
Hemoglobin
Myoglobin
Log of
Solubility
g mL-1
Fibrinogen
1
Serum
albumin
2
Molarity of AmSO4
3
1M
2M
AmSO4
3M
The concept of a Column
Ion Exchange Chromatography (IEC)
Separates molecules based on their charge
The side-chain groups of some amino acids are ionizable,
e.g., lysine, arginine, histidine, glutamic acid, aspartic acid
as are the N-terminal amino and C-terminal carboxyl groups
Thus proteins are charge molecules and can have a different charge
at a given pH because they have different compositions of ionizable
amino acids
For any given amphoteric protein, there will be a pH at which
its overall charge is 0
(No. of negative charges equals the No. of positive charges)
This is referred to as the ISOELECTRIC POINT (pI)
or ISOTONIC POINT of the protein
At a pH above its pI a protein will have a net negative charge
while
At a pH below its pI a protein will have a net positive charge
IEC resins are made by covalently attaching Negatively or Positively
charged functional groups to a solid support matrix to yield Cation
or Anion exchangers, respectively
Negatively charged exchangers bind positively charged ions – cations
Positively charged exchangers bind negatively charged ions – anions
At a pH=pI of a protein, it will not bind to an ion exchange
resin
When a charged molecule is applied to an exchanger of
opposite charge, it is absorbed, while neutral ions or ions of
the same charge are eluted in the void volume of the column
(the volume that is not bound). The bound protein displaces
the counterions
Adsorbed molecules are commonly eluted with salt (changes
ionic strength of the column buffer) or pH, which change the
affinities of the bound proteins for the exchanger
Gel Filtration Chromatography (GFC)
GFC (also Size Exclusion Chromatography, Molecular Sieve
Chromatography or Molecular Exclusion Chromatography)
Separates molecules based on their size (& shape)
It can also be used to determine the size and molecular weight
of a protein
Separation occurs due to the differential diffusion of various
molecules into gel pores in a porous matrix. For protein
purification, the matrix typically consists of porous beads
(with pores of a specific size distribution) of an inert, highly
hydrated gel
Largest MW comes off first
Separation is due to exclusion or inclusion from the gel
matrix
Small molecules diffuse into the gel pores, retarding their
flow through the column, while large molecules do not enter
the pores and are rapidly eluted from the column
Proteins elute from the column in order of decreasing
molecular weight
Common gel matrices are dextran, agarose and
polyacrylamide. These matrices are manufactured with
different degrees of porosity, and thus can fractionate
different size ranges of proteins
Other Purification Methods:
Affinity Chromatography:
Separates molecules based on specific interactions between the
protein of interest and the column matrix
E.g. Antibodies which bind Protein
Enzyme which binds a co-enzyme or inhibitor
A ligand is covalently bound to a solid matrix (usually agarose)
which is then packed into a chromatography column
When a mixture containing the protein of interest is applied to
the column, the desired protein is bound by the immobilised
ligands, while all other proteins in the mixture, which should
have no affinity for the ligand pass through and are discarded
Affinity chromatography
(with HIS-tagged proteins)
Affinity chromatography can be performed using a number of
different protein tags.
poly-hisitidine
The histidine tag is very short (6 His residues)
Should not alter the conformation of the tagged protein
Should not be involved in artificial interactions.
The poly-his tag binds to a nickel chelate resin
Eluted by 1.0 M imidazole
List of Other Methods:
Hydrophobic Interaction Chromatography:
-hydrophobic interactions under high salt
Chromatofocussing:
-Fractionates based on a pH gradient generated in an ionexchange column
HPLC:
-Similar to IEF and GFC, but uses pressure to increase
flow rates in columns with small particle size to increase
resolution of peaks
Methods for Assessing Protein Purity
SDS-PAGE – Commonest method, rapid and sensitive
SDS
sodium dodecyl sulphate
PAGE
polyacrylamide gel electrophoresis
Migration of a molecule
in a electric field
Separates materials
based on size
Isoelectric focusing (IEF) can also be used, which separates
proteins by charge differences (induced by a pH gradient)
2D gel electrophoresis – Combination of SDS-PAGE and IEF
Separates by charge in the first dimension and then by size in the
second dimension
Mass spectrometry
Protein Purification
Objectives
Purity
Stable
Cost
Time
Protein purification steps
Extraction
Cell breakage
chemical
physical
Debris removal
straining
centrifugation
filtration
Solubilization
Inclusion bodies
Urea
Inhibition of proteases
Preliminary concentration
ammonium sulfate precipitation
ultrafiltration
dialysis
Purification steps
Ion exchange
Affinity chromatography
Gel filtration
Preparative HPLC
Final step
Crystallization