Target: Purify a protein from

10g of tissue!!
Tissue extraction, clarification and
concentration

Purification schedule
Groundwork: sort out an assay and some
properties of the protein
Extraction: Find a technique that maximises the
amount of the target protein extracted
Clarification: Remove debris and unwanted cellular
organelles
Concentration: remove water and small metabolites
Initial purification: ion exchange or hydrophobic
interaction chromatography
Affinity: Focus on biospecificity
Polishing: Remove minor contamination

The contents of the cell

drawn to scale

Organ, tissue, molecules and atoms drawn to scale

Prostate tissue enrich in

transglutaminase IV

Cirrhostic liver:
Transglutaminase 2 is abundant in liver cells.
Reported to be responsible for fibrosis in
cirrhosis

Task A: Find a suitable protein

assay
Can detect functional
groups
e.g. lysine or aromatic
residues
Can detect the peptide
bond
The peptide bond has a
reducing centre which in
alkali conditions can reduce
divalent metal ions e.g.
Cu2+
Cu1+
The biuret reaction works
with peptides longer than 3
amino acids

protein assay
based upon the peptide bond
Biuret (sensitivity 1-20mg ml-1 )
Lowry (sensitivity 0.1-1.0mg ml-1 ).
Popular but time consuming. Adapted
methods can be used with detergents.
Bicinchonchinic acid (BCA) (sensitivity:
normal method 0.05-1.0mg ml-1 . micro
method 0.001-0.01 mg ml-1). Easy to use
adapted for use with microplates and
works with detergents.

The Biuret reaction:

The interaction of divalent copper ions with
the peptide bond

The interaction of Cu and BCA

+

Chelation of metal ions

Electron rich atoms (e.g. oxygen and
unprotonated nitrogen) can donate 2 electrons
into vacant orbitals into some metal ions.

This is called co-ordinate covalent bonding or

dative covalent bonding
The resultant bond is not as strong as a covalent
bond but it is sufficiently strong to retain the
metal ion.

Important examples in biology include:

EDTA chelation of metal ions.
Proteins can have reduced sulphur
from cysteine at their active site
or on their surface. Divalent heavy
metal ions will bind irreversibly and
moderate the functionality of the
sulphur groups. EDTA is used in buffers to
effectively remove damaging metal ions
from solution.
EF-Hand motif in protein structure
The functional group of aspartate is
oxygen rich. They donate electrons
To hold calcium ions in the protein
structure

Important examples in biology include:

Magnesium ions (Mg2+ chelated by ATP)

Haem group in haemoglobin

(Fe2+)

Magnesium ions in chlorophyll

Immobilised metal ion affinity chromatography (IMAC)

protein assay
based upon the attached functional groups
Proteins contain tyrosine and
tryptophan which absorb light
at 280nm. Blank a spec with
appropriate buffer and read
directly at 280nm use Beer
Lamberts Law or Std graph to
convert reading into protein
concentration values. Quick but
prone to give erroneous results
due to interfering substances
Bradford method (sensitivity
0.1-1.0mg ml-1 ). Uses
Coomassie blue dye to bind to
lysine residues. Quick and
inexpensive but cannot be used
with reducing agents or
detergents.

Extraction
At the start of a purification protocol
Find a suitable assay for the target protein
(quantitative and simple to use). Colourimetric,
radiometric, chromatographic or antibody
methods are available.
Use suitable protein assay (BCA, Bradford,
Lowry)
Proteins are differentially expressed in different
tissues.
Need to screen different tissues to use the tissue
with the highest activity

Task B: Basic parameters and

Extraction
Before embarking on chromatography
spend a bit of time checking
pH optima for activity and storage.
Temperature stability.
Inhibitor, activators and cofactor
requirements (e.g. metal ions)
Use of proteolytic inhibitors?

Proteolytic activity
Proteinase (endoprotease) cleave internal peptide
bonds.
Active site serine residue (-OH) e.g. chymotrypsin or
trypsin.
Active site sulphydryl residue (-SH) e.g. cathepsin or
papain.
Active site Aspartate (-COOH )
Metallo-protease II Zn2+ requirement
Peptidases (exopeptidases) cleave single amino acids
from either the N- or -C terminus of proteins.
Carboxypeptidase Y (from yeast)
Bovine carboxypeptidase A (Zn2+ requirement)
Leucine aminopeptidase

Proteolytic activity
Take crude extract of the tissue that the purification is to be used
and perform a proteolytic activity assay in the presence of
proteolytic inhibitors.
Serine proteinases inhibited by esterase inhibitors e.g.
Phenylmethylsuphonyl flouride (PMSF). Other inhibitors include
Soybean trypsin inhibitor.
Sulphydryl proteinase inhibitors inhibited by alkalyting agents
e.g. Iodoactetimide or N-ethylmaleimide. Other inhibitors include
Antipain or E-64.
Acid proteinase inhibitors e.g. pepstatin
Metallo-proteinases e.g. EDTA.
Determine which inhibitors work to prevent proteolytic activity
and at what concentration. Include the inhibitors in future
extraction buffers.

Task C: Extraction techniques

Having established an ideal starting buffer and pH it is

worth examining different extraction techniques to
improve the extraction yield.
Different techniques are required with different tissues.
A bye product of mechanical disruption is heat. To
reduce the damage to proteins caused by heat cool
the buffers and apparatus
Reduce foaming
Add proteolytic inhibitors
Plant tissue extracts may also require the presence of
anti oxidase inhibitors (e.g. vit C)

Extraction techniques for animal

and plant tissue
homogeniser

Mortar and pestle

blenders

Extraction

Polytron homogenisers
can be used with soft
(liver or kidney) or hard
fibrous tissues (Heart or
some malignant tissue)

Extraction of Bacterial cells

Bacterial cells can have a hard glycoprotein coating which makes

extraction by conventional techniques difficult.
Cell pellet put into the pressure cell are then subjected to intense
pressure by mechanical means or by using a pressured gas (N 2).
The pressure is released when the valve is opened. The cells are
disrupted by the sudden pressure drop and subjected to shear
forces as they are forced through the small exit hole.

Sonication for the disruption of

cells

A sonicator provides a focused output of high energy

sound waves that can create holes in cell membranes
which result in cellular disruption.
Bacterial cells may also need the presence of small
beads (Ballatini beads) to punch holes in the glycocalax
covering the outside of some bacterial cells.

Centrifugation
Particles suspended in a liquid will move at a rate
which depends upon: the applied force on a bench this force is very low
and is due solely to the earths gravity;
the density difference between particles and liquid
(remember that particles can sediment or float);
the size and shape of the particles;
the viscosity of the medium.
To separate materials it is sometimes necessary to
increase the gravitational force.

Centrifugation animation
http://sites.sinauer.com/cooper6e/

What happens to particles in the

course of centrifugation run

Clarification techniques

Proteins within the cell are concentrated in a reducing atmosphere. After

disruption the proteins are now dilute in an oxidising atmosphere.
Add reducing agents e.g. 5mM dithiothreitol or 5mM 2-mercaptoethanol.
Having broken up the cells check the pH as the disruption of some
organelles (lysosomes in animal cells and vacuoles in plant cells)
releases acid into the extract.
A eukaryotic cell extract is a mixture of nuclei, mitochondria, mixed
membrane micelles, clumps of broken cells and cytoplasmic proteins.
Differential centrifugation will fractionate the organelles.
2,000g for 5 min to pellet nuclei and cell clumps
13,000g for 20 min to pellet mitochondria (chloroplasts)
100,000g for 60mins to pellet plasma membrane, ER and Golgi
What is left is the particle free supernatant (PFS).
If the target protein is in the pellet fractions then purification can proceed
on this fraction (immediate enrichment).
If the target protein is in the PFS chromatographic techniques will be
required for purification.

Clarification techniques
In industry large volumes make the use of
a centrifuge an unlikely technique.
Ultra filtration through membranes of
decreasing pore size are used to filter out
the larger particles (0.2m will filter out
nuclei, bacteria and mitochondria).
Membrane filters down to >10,000 cut off
can be used

Ultrafiltration devices
Pressurised air or an inert
gas is used to provide the
filtration vector. Agitation
is
used
to
impede
macromolecules
from
polarising
on
the
membrane surface (a
variety of membranes
with different Mr cut offs
are
available)
and
reducing filtration speed.

Ultrafiltration

Lab scale ultrafiltration uses compressed N2 to

drive the liquid through the membrane.
Industrial scale ultrafiltration may use hollow
filter cartridges.

Tangential Ultrafiltration devices

The solution to be
processed is pumped
under pressure across an
ultrafiltration membrane
and then returned to the
original reservoir. The
solution is progressively
concentrated or purified
as solvent and micromolecules pass through
the membrane into a
separate filtrate vessel.

Ultrafiltration devices
In general ultrafiltration can be very slow
due to membrane fouling
Some proteins bind irreversibly to the
membrane surface
Can be useful in the later stages of a
purification protocol.