7 steps to protein purification
(adapted from: B. Osterlund and J.-C. Janson, Science Tools from
Pharmicia Biotech 2, 3 1997)
A. Prior to starting the purification.
1. What are the purposes and aims of the purification? Decide the use of the purified protein and how much is required.
2. What is known about the protein? Collect information about the chemical and physical properties of the protein from the literature and available sequence data.
3. Choose or develop assays for the protein. Establish fast and reliable methods for determining amount and activity of protein.
4. Select the source of the protein: select a source that contains high concentrations of the protein in a stable environment.
B. Purification
5. Extracting the protein: develop an efficient extraction procedure.
6. Structure a purification procedure: develop efficient capture, intermediate purification steps and polishing methods.
7. Establish optimum conditions for storage.
1. PURPOSES AND AIMS
- confirm sequence information
- identify sites of post translation modification
eg. -phosphorylation sites
-glycosylation sites
-lipid attachment sites
- enzyme characterization
-crystal structure
- produce antibodies
2. WHAT IS ALREADY KNOWN ABOUT THE PROTEIN?
This information will help in designing purification procedure.
- physical/chemical properties
-pI
-Mr
-subunit compostition, presence of disulfide bonds
-is it glycosylated? phosphorylated?
- native environment
-soluble or membrane bound
-secreted
-where in the cell is it found
-stability
-heat, acid lability
-sensitivity to proteases
3. CHOOSE/DEVELOP ASSAY
biological or enzyme activity
protein determination (eg Abs at 280 nm, Bradford assay,
Lowry assay)
Units of activity:
amount of product formed per unit time
1 international unit = 1 umol/min
Specific activity: activity/unit protein
eg
umol/min/mgm
Enrichment or purification factor
S.A. fraction/S.A. starting material
or S.A. fraction/S.A. preceding fraction
4. SELECT SOURCE OF MATERIAL
- concentration: choose tissue, organism with high
concentration
of target protein
- developmental stage does level of protein
change with development
- can protein be induced by hormones or nutritional condition
- use of expression system; note that posttranslational
modification
of protein may be different in expression system than in parent tissue
- subcellular localization: if target protein
is localized to a particular organelle, eg mitochondria,
nucleus,
etc than may want to start purification by isolating that
organelle
5. EXTRACTION OF PROTEIN
- release of protein from cell/solubilization of protein
i. mechanical disruption of cell
homogenization
french press
blenders
ultrasound
hypo osmotic lysis
ii. chemical disruption
organic solvents (less common)
detergents, particularly for solubilization of
membrane
proteins
gentle: triton X-100, deoxycholate
strong: sodiumdodecyl sulfate (SDS)
Once released form normal cellular environment proteins must be
protected
from:
- denaturation: control pH, osmolarity
- proteolysis: add protease inhibitors eg. Ca2+
chelators
to inhibit calcium dependent proteases, PMSF
(phenylmethylsulfonyl
flouride) a serine protease inhibitor, leupeptin, a
thiol
protease inhibitor.
-phosphatases: add phosphatase inhibitors; eg
Naflouride,
glyceraldehyde-3-phosphate (competes for phosphatase),
Na-orthovanadate,
inhibits protein tyrosine phosphatases
-oxidation: add reducing agents (betamercaptoethanol,
dithiothreitol)
to maintain SH groups and metal chelators to reduce oxidation of
protein
by metal ions.
Keep pretein COLD (4oC)
AVOID FOAMING
6. PURIFICATION
1. Capture
- concentrates target protein
- may remove from potentially harmful contaminants
- some initial purification - generally small
- capture step generally has high capacity, can deal with
large volumes and large amounts of protein, but low
resolving
power
Capture step frequently, but not necessarily,
exploits
differences in protein solubility
a. Salt fractionation: most commonly used
procedure is use of ammonium sulfate precipitation. Ammonium
sulfate
effectively competes with protein for water and water of hydration is
stripped
from surface of protein. Allows interaction between hydrophobic
gropes
on protein surface, causing protein to precipitate.
Because
proteins have different groups on surface different proteins
precipitate
at different concentrations of ammonium sulfate so some fractionation
may
be achieved. Also precipiated protein is concentrated in
pellet
and can be resuspended in a smaller volume, thus concentration is
achieved. Ammonium sulfate is highly soluble
allowing
solutions of high ionic strength can be prepared. Also it
stabilizes
many proteins.
Make sure you know how to use chart for preparing ammonium sulfate solutions of increasing concentration and how, for eg, how would you make a 35% to 50% ammonium sulfate cut.
b. Organic solvents (less used now-a-days)
may be used for selective precipitation of proteins
ethanol, acetone, butanol
c. isoelectric precipitation
many proteins are insoluble at their isoelectric pH and this
may be used to differentially precipitate proteins.
2. Intermediate Purification
ion exchange
reverse phase/hydrophobic interaction
gel filtration
Things to consider:
- Capacity, sample volume
- protein concentration
- yield vs purification
Resolving power - becomes more important as purification proceeds
and as remaining proteins become more and more similar in properties
High capacity, lower resolution methods at start , followed by
lower capacity, higher resolution at end.
3. Polishing
-gel filtration
-affinity chromatography
7. Storage of protein
-Stabililty
some proteins will be unstable, particularly at low
concentrations,
and will have a tendency to denature when in purified state.
Native
confirmation may in some cases be stabilized by keeping purified
protein
in 50% glycerol, or 1% serum albumin or
ammonium
sulphate.
Store proteins at -70 -80o C but avoid repeated freeze
thaw. Store in small portions
8. Tests for purity
- enrichment factor
- gel electrophoresis, single band on gel, may be
more than 1 band if protein is heteromeric.
- end group analysis, single amino terminal amino acid
if protein is monomeric.
a) What is maximum purification possible?
Depends upon what % of starting protein purified protein
represents.
Maximum purification factor = 100 / % of total
protein represented by target protein.
eg. If protein x = 0.1% of total protein maximum purification = 100 / 0.1 = 1000 fold
b) What % of total protein does purified protein represent?
% of total protein = 100 / purification factor
eg. If purification factor for a pure protein is 3000 then protein
=
100 / 3000 =0.033% of total starting protein.
Sample parameters and changes associated with various
purification
procedures
Trouble shooting
Most frequent problem is loss of enzyme activity.
Causes include: Methodological errors such as incorrect assay, incorrect buffer composition, impure reagents
or such things as : proteolysis, the presence of inhibitors or absence of activators or cofactors and precipitation of protein.
Notes: proteolysis can be checked by gel electrophoresis after each step of the purification, precipitates may be visable and may be detected as a decrease in protein recovery, lost activators or cofactors may be relocated by mixing aliquots of specific fraction with other fractions and testing for regain of activity.
|
|
|
|
| Salt fractionation | >1mg protein/ml | smaller volume, higher concentration, higher ionic strength |
| gel filtration | small volume, low viscosity | decrease concentration, possibly change in buffer |
| ion exchange | low ionic strength, correct pH | higher ionic strength and/ot chnage in pH, increased concentration |
| affinity chromatography | compatability with specific binding conditions | change in concentration and chnage in buffer |
| reverse phase chromatography | small volume | presence of organic solvents |