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 - + + - - -
+ + + - -
+ + - + - +

Optimal salt conc. for solubility

-
Weak attractive force - + +
- + + -
- +
+ + - - - +
- - +
+ + - - + - +
- + -
High Salt
Compete for water - ++ + +
-- -- + ++ + + - + - + - +-
+ +- - - -- - + + + -
- --
- -+-+ + + - - + +
+ +
+ - - - + -+-
-++- + - - -++ - + +
+ + + -++
+- + + - -- -+ + - + +
- -
-+
+ - +
-+-+-+ ++-+-
+ - + - -
+ +-
++ +- - +
+ +- + +++ - -+- +
- + - +
++- -
-
+ - + +- +
- - +-+- - -++- - - -- - ++++ ++++ + -- - -

Debye-Huckel Theory
 Ammonium sulphate precipitation
Hemoglobin Myoglobin

Log of
Solubility

g mL-1

Fibrinogen Serum
albumin
1 2 3
Molarity of AmSO4
1M 2M 3M
AmSO4
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 ion-
exchange 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