Transcript How do we purify proteins? GFP as model system to learn

How do we purify proteins?
BioTic
August 5, 2011
Hamline University
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Outline
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Protein over-production
Preparation of homogenate
Purification techniques
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Ion Exchange Chromatography
Affinity Chromatography
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Think/Pair/Share
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What would you need to consider
before starting a protein purification?
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Name at least three important points to
consider
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Before starting a purification….
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Have a good source of the protein of
interest
Knowledge about protein composition
and properties
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Why?
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It will help you decide what purification
technique will work best
Activity assay to follow the purification
of the protein
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Steps for Protein Purification
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Isolation of the gene of interest
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Transfer the gene of interest to suitable organism
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Transformation
Protein production
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cloning
Induction of promoter that controls the gene of interest
Cell Lysis
Purification
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How do we choose a purification method?
What are the common methods of Protein purification?
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Getting Enough Protein
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For protein purification a “rich source” of
the protein of interest is needed
Can you name some common sources:
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Tissues
Recombinant Cell Extracts
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Recombinant Cell Extracts
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Genes encoding the protein
of interest are often cloned
in bacteria (E.coli) or yeast
to facilitate over-production
Microbial cells are easy to
grow and manipulate
Cells lysis is simple making
protein extraction fast and
convenient
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Production of Recombinant Proteins
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How are we over-producing GFP?
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Beta Lactamase
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Green Fluorescent
Protein (GFP)
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Ampicillin resistance
Aequorea victoria
jellyfish gene
araC regulator
protein
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Regulates GFP
transcription
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We have cells over producing
protein…. What is next?
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Getting the Protein out the Cells:
Preparation of Homogenates
Break cells,
tissue, or
organ
Blender,
homogenizer,
sonication,
Microbial cells
pressure,
or tissue
psmotic
Supernatant with
Soluble protein
Pellet with intact cells, organelles,
membranes and membrane proteins
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Separating the Protein of Interest
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Most common technique
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Ammonium sulfate precipitation
Also known as “salting proteins out”
Principle
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Ammonium sulfate is very polar
Binds water molecules easily
Competes with proteins for water molecules
Eventually proteins lose and “salt out”
(precipitate)
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Ammonium Sulfate Precipitation
• Fully hydrated
• Solubility depends on
Interactions with water molecules
• Ammonium sulfate molecules
interacting with water molecules
( taking over)
• Proteins fall out of solution
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Further Purification
Chromatography
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Chromatographic separation of proteins
are accomplished by several
techniques, the most common are:
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Ion Exchange Chromatography ( IEX)
Affinity Chromatography
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Ion Exchange Chromatography
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Principle
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Column filled with a charged resin
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Negatively charged column ( cation exchanger)
Positively charged column ( anion exchanger)
Amino acids will bind to the resin according
to their ionization state
The ionization state of the amino acids will
be determined by the pH of the mixture
and the pH of the elution buffer
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Ion Exchange Chromatography
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Affinity Chromatography
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Principle
 Interactions between the protein of interest and a ligand
bound to a gel matrix
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Compounds used for affinity chromatography are
usually recognized by the protein of interest
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Lactate , pyruvate and NAD+ are recognized by lactate
dehydrogenase
Nickel and histidine tag proteins
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How are proteins eluted ?
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Most affinity and ionic exchange columns are
eluted using high salt
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Why?
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The salt ions compete with the protein for the charges in the
column
High salt conditions destroy electrostatic interactions holding
the protein to the column
Affinity columns can be eluted with a substrate or
compound that the protein recognizes and can
compete with the affinity ligand
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Laboratory Exercise #5
GFP Purification
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GFP-Properties
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Isolated from from Jelly FishAquoera Victoria
Function
 To absorb blue light from
another jelly fish protein
aequorin
 Upon absorption of blue
light, the GFP emits green
light
 Absorption at 385 nm and
emission is at 485 nm
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
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Qui ckTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this pi cture.
GFP Story
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Qui ckTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this pictur e.
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Nobel prize in chemistry,
2008
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Osamu Shimomura,
Martin Chalfie
Roger Y.Tsien
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Qui ckTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this pi cture.
Quic kTime™
and a and a
QuickTime™
(Uncompressed) decompressor
TIFFTIFF
(Uncompressed)
are needed todecompressor
see this picture.
are needed to see this picture.
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GFP Structure
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11 beta sheets
Cylindrical shape
The fluorophore is in
the center of the
structure
Known as the “paint
can”
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GFP Fluorophore
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Originates from an internal ser-tyr-gly sequence
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Cyclization and post-translational modification of of this tri-peptide
allows the formation of a ring system which is resonant and produces
fluorescent upon excitation
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GFP Fluorophore
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GFP Art
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
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GFP Purification
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Method
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Hydrophobic interaction Chromatography
Why?
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GFP has long stretches of hydrophobic amino
acids in its inner core
Upon exposure of the protein to binding buffer, a
conformational change occurs and the
hydrophobic region is exposed
Exposure of the hydrophobic region allows binding
of GFP to the hydrophobic resin in the column
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HIC Macro-Prep Column
(Biorad)
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Methylco-polymer support (beads)
In the presence of high salt the hydrophobic groups
of the protein interact with the methyl groups in the
resin allowing binding
Washing with “low salt” buffer allows the less
hydrophobic proteins to wash out
Final wash with “non-salt buffer”, abolishes
hydrophobic interactions and allows the hydrophobic
protein to be elute off the column
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Hydrophobic Interactions
Hydrophobic
bead
O
-O
S
O-
O
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Hydrophobic Interaction
Chromatography
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Add bacterial lysate to column matrix in
high salt buffer
2. Wash less hydrophobic proteins from column in low
salt buffer
3. Elute GFP from column with no salt buffer
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Hydrophobic interactions
Step #1
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Add bacterial
lysate to column
matrix in high
salt buffer
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Hydrophobic
proteins interact
with column
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Step #2
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Wash less
hydrophobic from
column with low
salt buffer
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Less hydrophobic
E. coli proteins fall
from column
GFP remains
bound to the
column
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Step#3
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Elute GFP from
column by
adding no salt
buffer
GFP
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Released from
column matrix
Flows through
the column
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Food for thought
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How would you determine if your GFP
purification was effective?
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Propose two experiments
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