Green Fluorescent Green Fluorescent Protein: A Reporter Protein: A Reporter Molecule Molecule 1. 1. Transformation of pGLO Transformation of pGLO plasmid plasmid 2. 2. Purification of GFP Purification of GFP 3. 3. PAGE Analysis of Purified PAGE Analysis of Purified
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Green Fluorescent Protein: A Reporter Molecule 1. Transformation of pGLO plasmid 2. Purification of GFP 3. PAGE Analysis of Purified GFP (if we have time)
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Green Fluorescent Protein: Green Fluorescent Protein: A Reporter MoleculeA Reporter Molecule
1.1. Transformation of pGLO plasmidTransformation of pGLO plasmid
2.2. Purification of GFPPurification of GFP
3.3. PAGE Analysis of Purified GFP (if we PAGE Analysis of Purified GFP (if we have time)have time)
Transformation and Purification of Green Fluorescent Protein (GFP)
Central Framework of Molecular Central Framework of Molecular BiologyBiology
GFP is a visual markerGFP is a visual marker
Study of biological processes Study of biological processes (example: synthesis of proteins)(example: synthesis of proteins)
Localization and regulation of gene expressionLocalization and regulation of gene expression
Cell movementCell movement
Cell fate during developmentCell fate during development
Formation of different organsFormation of different organs
Screenable marker to identify transgenic organismsScreenable marker to identify transgenic organisms
DNA RNA Protein Trait
Where Does It Come From?Where Does It Come From?
Aquatic origin Aquatic origin Aequorea victoriaAequorea victoria About 120 light About 120 light
emitting organs emitting organs Means of visual Means of visual
communicationcommunication PredationPredation MatingMating SymbiosisSymbiosis Warning signalWarning signal
GFP Structure – The Beta BarrelGFP Structure – The Beta Barrel
238 amino acids238 amino acids Cylindrical foldCylindrical fold Very stable structure Very stable structure
that is resistant to that is resistant to denaturing denaturing
Alpha helices are red and beta pleated sheets
are green.
GFP’s ChromophoreGFP’s ChromophoreChromophores are also called fluorophores!Chromophores are also called fluorophores!
Composed of Composed of Ser-Ser-Gly-Tyr Gly-Tyr amino acid amino acid sequencessequences
Oxygenating the Oxygenating the molecule helps it to molecule helps it to fluorescefluoresce under a under a ‘black light’‘black light’
Why a Black Light?Why a Black Light?
an influx of Ca+2 causes an influx of Ca+2 causes the first protein, aequorin, the first protein, aequorin, to become excited and to become excited and transfer the energy to the transfer the energy to the second protein, GFP, second protein, GFP, which loses the energy by which loses the energy by emitting a photon of emitting a photon of green light green light
The flurophore is The flurophore is embedded in the beta embedded in the beta barrel structurebarrel structure
Absorbs light at 395 Absorbs light at 395 and 470 nm and emits and 470 nm and emits light at 509 nm (light at 509 nm (green green light)light)
In the OrganismIn the Organism
GFP As A Biological TracerGFP As A Biological Tracer
The Nobel Prize in 2008The Nobel Prize in 2008
In 2008, Osamu Shimomura, Marty Chalfie In 2008, Osamu Shimomura, Marty Chalfie and Roger Tsien won the Nobel Prize in and Roger Tsien won the Nobel Prize in chemistry for isolating GFP and using it as chemistry for isolating GFP and using it as a a ‘reporter molecule’‘reporter molecule’ in biotechnology. in biotechnology.
Osamu Shimomura Martin Chalfie Roger Tsien
What’s a Reporter Molecule?What’s a Reporter Molecule?
A A reporter moleculereporter molecule is is one protein (like GFP) one protein (like GFP) linked to the protein linked to the protein you are interested in you are interested in studying.studying.
You can follow what You can follow what your protein is doing your protein is doing by following the by following the reporter molecule reporter molecule (GFP).(GFP).
The LabThe Lab
There are 3 parts to this laboratory!There are 3 parts to this laboratory!
Transformation of pGLO plasmidTransformation of pGLO plasmid Purification of GFPPurification of GFP
PAGE Analysis of Purified GFPPAGE Analysis of Purified GFP
General Transformation ProcedureGeneral Transformation Procedure
Transformation ProcedureTransformation Procedure Suspend bacterial colonies in Transformation solutionSuspend bacterial colonies in Transformation solution
Add pGLO plasmid DNAAdd pGLO plasmid DNA
Place tubes on icePlace tubes on ice
Heat-shock at 42°C and place on iceHeat-shock at 42°C and place on ice
Incubate with nutrient brothIncubate with nutrient broth
Medium that contains Medium that contains nutrients for bacterial nutrients for bacterial growth and gene growth and gene expressionexpression CarbohydratesCarbohydrates Amino acidsAmino acids NucleotidesNucleotides SaltsSalts VitaminsVitamins
1.1. TransformationTransformationUptake of foreign DNA, often a circular plasmidUptake of foreign DNA, often a circular plasmid
Transform Transform the pGLO the pGLO plasmid into plasmid into E. coliE. coli
Be sure to follow the Be sure to follow the directions…exactly directions…exactly as they appear in the as they appear in the protocol.protocol.
2. 2. Preparation for Purification of Preparation for Purification of GFPGFP
1.1. Make +pGLO Make +pGLO cultures cultures
2.2. Aerate Aerate
3.3. Equilibrate HIC Equilibrate HIC beads & prepare a beads & prepare a tube of HIC resin tube of HIC resin
Lecture #2Lecture #2
3. 3. Purification of GFPPurification of GFP
Purify GFP using Purify GFP using hydrophobic interaction hydrophobic interaction chromatography (HIC)chromatography (HIC)Lyse GFP cellsLyse GFP cells Incubate in high-salt binding bufferIncubate in high-salt binding buffer
This turns the GFP molecule inside out to reveal This turns the GFP molecule inside out to reveal hydrophobic chromophorehydrophobic chromophore
GFP chromophore binds to HIC resinGFP chromophore binds to HIC resinRelease GFP from resin and restore structureRelease GFP from resin and restore structureView View fluorescencefluorescence
Why Use HIC?Why Use HIC?
To purify a single recombinant To purify a single recombinant protein of interest from over protein of interest from over 4,000 naturally occurring 4,000 naturally occurring E. E. colicoli gene products. gene products. AKA…to get lots of pure AKA…to get lots of pure
1.1. Add bacterial lysate to column matrix in Add bacterial lysate to column matrix in high salt bufferhigh salt buffer
2.2. Wash less hydrophobic proteins from Wash less hydrophobic proteins from column in low salt buffercolumn in low salt buffer
3.3. Elute GFP from column with no salt Elute GFP from column with no salt bufferbuffer
Step 1 – HICStep 1 – HIC
Add bacterial lysate to Add bacterial lysate to column matrix in column matrix in high high salt buffersalt buffer Hydrophobic proteins Hydrophobic proteins
interact with columninteract with column Salt ions interact with Salt ions interact with
the less hydrophobic the less hydrophobic proteins and Hproteins and H22OO
Hydrophobic bead
NH
H
H+
H
-+
+
O
S
O
O O- -
O
S
OO
O
-
-NH
H
H+
H
-+
+
O
S
O
O O- -
O
S
OO
O
-
-
Step 2 - HICStep 2 - HIC Wash less Wash less
hydrophobic from hydrophobic from column with column with low salt low salt bufferbuffer Less hydrophobic Less hydrophobic
E. coli proteins fall E. coli proteins fall from columnfrom column
GFP remains bound to GFP remains bound to the columnthe column
NH
HH+
H
-+
+
O
S
O
O O- -
O
SO
O
O
-
-
Hydrophobic bead
-+
+
-+
+-+
+
Step 3 - HICStep 3 - HIC
Elute GFP from Elute GFP from column by adding a column by adding a
no-salt bufferno-salt buffer
GFPGFP Released from column Released from column
matrixmatrix Flows through the Flows through the
columncolumn
Hydrophobic bead
-
+
+-+
+ -
+
+
-+
+
-
+
+
GFP PurificationGFP Purification
Day 1 Day 2
Day 3
Helpful HIC HintsHelpful HIC Hints Add a small piece of paper to Add a small piece of paper to
collection tube where column collection tube where column seats to insure column flowseats to insure column flow
Rest Rest pipet tip on side of column to avoid pipet tip on side of column to avoid column bed disturbance when adding column bed disturbance when adding solutionssolutions
Drain Drain until the meniscus is until the meniscus is justjust above the matrix for best separationabove the matrix for best separation