Download Green Fluorescent Protein: A Reporter Molecule

Green Fluorescent Protein:
A Reporter Molecule
1.
3.
Transformation of pGLO plasmid
2. Purification of GFP
PAGE Analysis of Purified GFP (if we
have time)
Transformation and Purification of
Green Fluorescent Protein (GFP)
Central Framework of Molecular
Biology
DNA
RNA
Protein

GFP is a visual marker

Study of biological processes
(example: synthesis of proteins)

Localization and regulation of gene expression

Cell movement

Cell fate during development

Formation of different organs

Screenable marker to identify transgenic organisms
Trait
Where Does It Come From?



Aquatic origin
Aequorea victoria
About 120 light
emitting organs

Means of visual
communication




Predation
Mating
Symbiosis
Warning signal
GFP Structure – The Beta Barrel



Alpha helices are red
and beta pleated sheets
are green.
238 amino acids
Cylindrical fold
Very stable structure
that is resistant to
denaturing
GFP’s Chromophore
Chromophores are also called fluorophores!

Composed of SerGly-Tyr amino acid
sequences

Oxygenating the
molecule helps it to
fluoresce under a
‘black light’
Why a Black Light?

The flurophore is
embedded in the beta
barrel structure

Absorbs light at 395
and 470 nm and
emits light at 509 nm
(green light)
In the Organism

an influx of Ca+2 causes
the first protein, aequorin,
to become excited and
transfer the energy to the
second protein, GFP,
which loses the energy by
emitting a photon of
green light
GFP As A Biological Tracer
The Nobel Prize in 2008

In 2008, Osamu Shimomura, Marty Chalfie
and Roger Tsien won the Nobel Prize in
chemistry for isolating GFP and using it as
a ‘reporter molecule’ in biotechnology.
Osamu Shimomura
Martin Chalfie
Roger Tsien
What’s a Reporter Molecule?

A reporter molecule is
one protein (like GFP)
linked to the protein
you are interested in
studying.

You can follow what
your protein is doing
by following the
reporter molecule
(GFP).
The Lab
There are 3 parts to this laboratory!


Transformation of pGLO plasmid
 Purification of GFP
PAGE Analysis of Purified GFP
General Transformation Procedure
Transformation Procedure

Suspend bacterial colonies in Transformation solution

Add pGLO plasmid DNA

Place tubes on ice

Heat-shock at 42°C and place on ice

Incubate with nutrient broth

Streak plates
Why Perform These Steps?
Transformation solution
= CaCI2
1.
Ca++
O
++
O P O
CaBase
O
CH2
Base
O
Sugar

Positive charge of Ca++
ions shields negative
charge of DNA
phosphates
Ca++
Ca++
O
O P O
Base
O
CH2
O
Sugar
OH
Why Perform These Steps?
2. Incubate on ice slows fluid cell membrane
3. Heat-shock Increases permeability of membranes
4. Nutrient broth incubation Allows beta-lactamase (amp resistance)
expression
What’s LB?

Luria-Bertani (LB) broth

Medium that contains
nutrients for bacterial
growth and gene
expression





Carbohydrates
Amino acids
Nucleotides
Salts
Vitamins
1. Transformation
Uptake of foreign DNA, often a circular plasmid
GFP
Transform the pGLO
plasmid into E. coli
Be sure to follow the
directions…exactly
as they appear in the
protocol.
Beta-lactamase
Ampicillin
pGLO plasmids
Resistance
Transcription Regulation

Lactose
operon

Arabinose
operon

pGLO
plasmid
Transcriptional Regulation
Effector = Regulatory Molecule
ara Operon
lac Operon
LacI
Z
Y A
ara
C
Z
Y A
araC
Y A
B A D
RNA Polymerase
RNA Polymerase
Z
A D
Effector (Arabinose)
Effector (Lactose)
LacI
B
araC
B A D
Gene Regulation
ara GFP Operon
ara Operon
ara
C
B
A D
araC
Effector (Arabinose)
Effector (Arabinose)
araC
B A D
araC
RNA Polymerase
araC
B A D
GFP Gene
GFP Gene
RNA Polymerase
araC
GFP Gene
2. Preparation for Purification of
GFP
1.
2.
3.
Make +pGLO
cultures
Aerate
Equilibrate HIC
beads & prepare a
tube of HIC resin
Lecture #2
3. Purification of GFP

Purify GFP using hydrophobic interaction
chromatography (HIC)
Lyse GFP cells
 Incubate in high-salt binding buffer

 This
turns the GFP molecule inside out to reveal
hydrophobic chromophore
GFP chromophore binds to HIC resin
 Release GFP from resin and restore structure
 View fluorescence

Why Use HIC?

To purify a single recombinant
protein of interest from over
4,000 naturally occurring E.
coli gene products.

AKA…to get lots of pure
product!
Hydrophobic Interaction
Chromatography
The Steps
1.
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
Step 1 – HIC

Add bacterial lysate to
column matrix in high
salt buffer


Hydrophobic proteins
interact with column
Salt ions interact with
the less hydrophobic
proteins and H2O
Hydrophobic
bead
Step 2 - HIC

Wash less
hydrophobic from
column with low salt
buffer
Less hydrophobic
E. coli proteins fall
from column
 GFP remains bound to
the column

O
- O S OO
Hydrophobic
bead
Step 3 - HIC

Elute GFP from
column by adding a
no-salt buffer
GFP


Released from column
matrix
Flows through the
column
Hydrophobic
bead
GFP Purification
Day 3
Day 1
Day 2
Helpful HIC Hints

Add a small piece of
paper to collection
tube where column
seats to insure
column flow

Rest pipet tip on side of
column to avoid column bed
disturbance when adding
solutions

Drain until the meniscus is just
above the matrix for best
separation
4. PAGE electrophoresis
SDS Page

SDS PAGE sample
preps are made from
white and green
colonies

Bacterial lysates are
prepared in Laemmli
buffer

Samples are loaded
onto polyacrylamide
gels
LB/amp
LB/amp/ara
GFP Visualization-During & Post
Electrophoresis



Samples are
electrophoresed
MWG
MWG
M W G
Fluorescent GFP can
be visualized during
electrophoresis
Fluorescent
isoform
Coomassie stained
gels allow for
visualization of
induced GFP proteins
Non-fluorescent
isoform
During Electrophoresis
Prestained bands
+ UV activated GFP
Fluorescent
bands
Post Electrophoresis
Coomassie stained
bands
Any Questions?