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

Streak platesStreak plates

Why Perform These Steps?Why Perform These Steps?

1.1. Transformation solution Transformation solution = CaCI= CaCI22

Positive charge of Ca++ Positive charge of Ca++ ions shields negative ions shields negative charge of DNA charge of DNA phosphatesphosphates

Ca++

Ca++

OCH2

O

P O

O

OBase

CH2

O

P

O

O

O

OH

OCa++

Sugar

Sugar

Base

Base

Ca++

Why Perform These Steps?Why Perform These Steps?

2. 2. Incubate on iceIncubate on ice - - slows fluid cell membraneslows fluid cell membrane

3. 3. Heat-shockHeat-shock - - Increases permeability of membranesIncreases permeability of membranes

4. 4. Nutrient broth incubationNutrient broth incubation - - Allows beta-lactamase (amp resistance)Allows beta-lactamase (amp resistance)expressionexpression

What’s LB?What’s LB?

LLuria-uria-BBertani (LB) brothertani (LB) 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.

GFP

Beta-lactamase

Ampicillin

ResistancepGLO plasmids

Transcription RegulationTranscription Regulation

Lactose Lactose operonoperon

Arabinose Arabinose operonoperon

pGLO pGLO plasmidplasmid

Transcriptional RegulationTranscriptional Regulation

B A DaraC

B A DaraC

RNA Polymerase

Effector (Arabinose)

araC B A D

ara Operon

RNA Polymerase

Z Y A

Z Y ALacI

Effector (Lactose)

Z Y ALacI

lac Operon

Effector = Regulatory Molecule

Gene RegulationGene Regulation

RNA Polymerase

araC

ara GFP Operon

GFP Gene

araC GFP Gene

araC GFP Gene

Effector (Arabinose)

B A DaraC

B A DaraC

RNA Polymerase

Effector (Arabinose)

araC B A D

ara Operon

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

product!product!

Hydrophobic Interaction Hydrophobic Interaction ChromatographyChromatography

The StepsThe Steps

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

4. PAGE electrophoresis4. PAGE electrophoresis

SDS PageSDS Page SDS PAGE sample preps SDS PAGE sample preps

are made from white and are made from white and green colonies green colonies

Bacterial lysates are Bacterial lysates are prepared in Laemmli prepared in Laemmli bufferbuffer

Samples are loaded onto Samples are loaded onto polyacrylamide gels polyacrylamide gels

LB/amp LB/amp/ara

GFP Visualization-During & Post GFP Visualization-During & Post ElectrophoresisElectrophoresis

Samples are Samples are electrophoresed electrophoresed

Fluorescent GFP can Fluorescent GFP can be visualized during be visualized during electrophoresis electrophoresis

Coomassie stained Coomassie stained gels allow for gels allow for visualization of visualization of induced GFP proteins induced GFP proteins

During Electrophoresis Post Electrophoresis

M W GM W GM W G

Fluorescent isoform

Non-fluorescent isoform

Prestained bands+ UV activated GFP

Fluorescentbands

Coomassie stainedbands

Any Questions? Any Questions?