Download Part I- Protein Purification

Part I- Protein Purification
Each protein folds into a specific shape and
has a unique 3-D conformation.
Objectives of Protein Purification Step:
• To separate one protein from a complex mixture of
proteins (such as a tissue extract), while maintaining
Biological Function.
• One can maintain biological function by controlling the
pH, the temperature and the ionic strength (ie. salt
concentration).
Considerations in Protein Purification
A. Selection of Protein Source
• From natural source
• From E. Coli host cells through molecular cloning
• From Yeast host cells through molecular cloning
• From mammalian cells or insect cells through transfection
B. Protein solubility and stability
• obtain crude protein extracts from host cells
• stabilization of proteins
* Native State: adjust pH, ionic strength, temp, buffer
solution constituents, reducing reagent etc.
* denature Æ renature
C. Assay of proteins
• unique assay for protein of interest(For an enzyme, its biological activity is used as a unique
assay for its presence in a tissue extract.)
• Assay for total protein and its’ purity.
• functional assay
D. Quantity (How Much?), Quality (How Pure?)
eg. Substance of < 0.1% tissue dry weight Æ 98% pure
Protein Purification Strategies
To purify the protein you use methods based on differences
in:
1. Surface charge on proteins
2. Molecular size of protein (related to molecular
weight)
3. Biological activity due to binding to substrates or
inhibitors)
4. Other chromatographic methods (reverse-phase,
hydrophobic interaction etc.)
Ion exchange chromatography
The surface charge, or the net charge, of the protein at a set pH will be
negative, neutral, or positive which depend on the pI of the protien. Thus,
one can use ion exchange chromatography to separate proteins with
differing charges.
Principles of ion exchange chromatography
Ion exchange chromatography
Ion exchange chromatography
১
ம
(gradient)
Gel Filtration Chromatography
Also called Size exclusion chromatography, The
size of protein is related to its’ molecular
Weight. Thus, proteins can be separated like
particles in a sieve-done with gel beads.
Gel Filtration Chromatography
Gel bead has molecular size holes so that small molecules like water and buffer enter it
completely. Some proteins are small enough to also enter the molecular holes in the Gel bead.
Other proteins are too large to enter holes and pass by the Gel bead.
In Gel Filtration, larger proteins elute first, medium sized ones next and finally
smallest proteins elute last.
Standardization of gel filtration experiment
(1) Determine the
molecular weight of
the target molecule.
(2) Purification
(3) Estimate the
polymerization state
of the molecules.
Vo: void volume
Ve: the volume of solvent required to elute solute (protein etc) from the column.
Ve/Vo: relative elution volume, which is independent of the size of the particular column used.
Affinity Chromatography
Affinity chromatography
depends on the biological
activity or the affinity of the
protein molecule for its
substrates. It is usually a very
effective method of purification.
Sometimes, a protein can be
purified to homogeneity
(complete purity) using only
affinity chromatography.
Affinity Chromatography
Purify monoclonal rat IgG using affinity chromatography
Lan1: rat hybridoma cell culture fluid, dilute 1;10
Lane2: flow through, dilute 1:10
Lane3: purified monoclonal rat IgG, dilute 1:5
Cross-linking of functional group
Extension of functional group
Coupling other groups
(1)
(2)
(3)
(4)
spacer arm may be extended by
reaction with succinic anhydride (VI)
Phenolic group may be attached via
diazonium derivatives(VII) or via
bromoacetamidoalkyl derivative(V)
Carboxyl group are coupled by
carbodiimide reaction(III)
Thiol derivative(VI) couple carboxyl
groups in the presence of
carbodiimide and the thiol ester bond
may be cleaved using hydroxylamine.
Affinity Chromatography Application
Straviden + biotin
Streptavidin is a protein which acts like an antibiotic by
removing biotin from the surrounding environment and
inhibiting the growth of competitive microorganisms. The
molecular weight of native Streptavidin is ~ 130,000 daltons
(octameric protein), the molecular weight of the monomer is ~
16,000 daltons. Streptavidin, like avidin, specifically binds the
vitamin biotin. Unlike avidin, Streptavidin is not glycosylated
and shows significantly less non-specific binding. KD=10-15 M
Affinity Chromatography Application
ҔٰપϯDNA-binding protiens
კҢ΋ࢤ‫ڀ‬Ԗbinding site‫ޑ‬DNAТ
ࢤǴӧ‫׀‬ᆄೱ΢biotinylated nucleotide.
࿶ᆶೈқ፦mixtureϸᔈࡕǴӆу΢
tetrameric streptavidinǴౢғternary
complexǴ࿶җbiotin-celluloseᐋિ֎
ߕǴԶ‫ځ‬дූᎩೈқ፦೏ؑࢱǴҞ኱
ೈқ፦ӆ࿶ଯᡶྋనϩᚆрٰǶ
Affinity Chromatography Application
GST(glutathio-S-transferase) catalyzes the
addition of the glutathion sulfhydryl group to
the epoxide, forming the first
peptidoleukotrienes, leukotriene C4 (LTC4).
Leukotrienes are involved in various
inflammatory disorder and asthma.
J-glutamylcysteinylglycine,
glutathione (GSH)
GST pull down purification
ճҔaffinity chromatography‫ޑ‬
চ౛Ǵёаપϯ‫”܈‬ഄр”ᆶ
target proteinϕ࣬ϸᔈ‫ޑ‬ғϯϩ
ηǶ
bait-protein: prepared as GST fusion protein
Test protein: either purified or labeled by in- vitro
translation.
Metal Chelate Affinity Chromatography
MetGlySerSerHisHisHisHisHisHisSerSerGlyLeuValProArgGlySer....
Example of a recombinant His-tag protein sequence
Hydrophobic Interaction Chromatography (HIC)
Reverse Phase Chromatography (RPC)
The resin used in hydrophobic
interaction method
CH3
-O-Si-CH2-CH2-CH2-CH3
CH3
C4 column
CH3
-O-Si-(CH2)17-CH3
CH3
C18 column
CH3
-O-Si-(CH2)7-CH3
CH3
C8 column
Agarose-octyl group
Agarose-Phenyl group
Reverse Phase Chromatography
Dialysis )೸‫*݋‬
Purpose:
• Reduce ionic strength of the solution
• Concentrate protein sample
SUMMARY of PURIFICATION PROCEDURES
To obtain a homogeneous protein/enzyme, one must apply a combination
of the methods.
For example, ion-exchange chromatography is applied first, which
separates proteins by difference in ionic properties;
Then gel filtration is applied, which separates by difference in molecular
size. Since few proteins have exactly the same ionic character and same
molecular size, a high degree of purity will be achieved by a combination
of these two purification methods.
Affinity chromatography, which is much more specific for a particular
enzyme, will in the end usually render an enzyme homogeneous.