Transcript What is a protein?

Protein Purification
with fluorescent proteins
Organisms produce thousands of different proteins,
each having a different function…
Hair and Nails
Structural Support
Enzymes
Antibodies
Hormones
Muscle Contraction
Receptors,
membrane channels
Nutrient Storage
Protein Structure
1° = amino acids
2° = basic structure
(hydrogen bonds)
3° = 3D structure
4° = interaction
of subunits
Protein Structure
Proteins…
…are created by living organisms
(DNA → RNA → PROTEIN → trait)
…have unique structures that determine function
(insulin, cobratoxin, fluorescence)
…can be isolated from living things
(humans, cobras, jellies)
…can be studied and modified by humans
(fluorescent proteins)
Engineered Fluorescent Proteins
From organism…
…to purified protein product
Making a Protein in the Lab
“Transformation”
Bacterial genome
Plasmid
E. coli bacterial cell
We use E. coli bacteria “transformed”
with a plasmid (a loop of DNA).
Making a Protein in the Lab
DNA
RNA
Protein
E. coli bacterial cell
The bacteria now express (make)
the fluorescent protein.
Our Plasmids
Plasmid Mix 1
Plasmid Mix 2
GFP
Cherry
FP gene
BFP
Tangerine
Grape
YFP
AmpR
Ampicillin
resistance gene
“Ampicillin resistance”
means the bacteria
will survive even when
exposed to antibiotics!
Why Purify Proteins?
Research
•
to understand structure
Medicine
•
to make vaccines
•
to treat disorders
How do you purify proteins?
Purify a specific protein from over 4,000
naturally occurring E. coli gene products.
How do you purify proteins?
Fluorescent proteins are
just one of thousands of
proteins in the cell!
Fluorescent Protein
How do you purify proteins?
1. Open the cells
2. Separate cell components
3. Distinguish the protein of interest
4. Separate the protein of interest
5. Retrieve the protein of interest
“Column Chromatography”
Ni2+
Lysozyme
Lysozyme is a naturally occurring enzyme
that is used to break open cells.
How do you purify proteins?
“Snap Freeze” Cell Lysis
Freezing and then thawing…
…causes ice crystals to break the cell open.
Purpose of the Nickel Beads
The “his tag” is
how the protein
attaches to the
“nickel bead”!
Fluorescent Protein
with “his tag”
The “his tag”
Purpose of the Nickel Beads
The nickel bead binds to the “his tag”
of the fluorescent protein.
Ni2+
Joined together, the
FP and nickel bead
are too BIG to pass
through the cotton!
How do you purify proteins?
Fluorescent Proteins
The nickel beads are too
BIG to pass through the
column, so the FPs that
are stuck to nickel beads
stay on top of the cotton.
All other proteins will flow
through the cotton ball into
the waste tube.
How do you purify proteins?
Elution
Imidazole
FP are separated from
nickel beads by the
imidazole (elution buffer).
Histidine
Now FP is small
enough to pass
through the column.
Ni2+
Protocol Summary
1. Lyse (cut) open the cells.
2. Centrifuge to create pellet.
Ni2+
3. Mix supernatant with nickel beads.
4. Pass the supernatant through the column.
5. Add elution buffer.
6. End with a pure sample containing
only the fluorescent protein.
Roger Tsien and Rainbow Proteins
Roger Tsien and Rainbow Proteins
GFP
RFP
Fluorescent Organisms
Corals
Jellyfish
Amphipod
Spider’s palps
The “Central Dogma”
DNA
mRNA
Protein
Trait
Engineered Fluorescent Proteins
From GFP:
1. Green
2. Blue
3. Grape
We have six
different plasmids!
From RFP:
4. Cherry
5. Tangerine
6. Yellow
Laboratory Introduction
What is a protocol?
What is a protein?
Why do scientists
use protocols?
Why would we need to
purify proteins?
Now let’s
practice
pipetting!
Protein Structure
Structure
determines
function.
Now you be
the protein!
Why Purify Proteins?
Pancreas
→
Identify Cells
→ Insert Plasmid into Cell
→
Isolate Gene
→ Cell Creates Insulin
→ Human Use
→
Insert Gene
→ Isolate/Purify Protein
Insulin for
diabetics
Why Purify Proteins?
Pancreas
→
Identify Cells
→ Insert Plasmid into Cell
→
Isolate Gene
→ Cell Creates Insulin
→ Human Use
→
Insert Gene
→ Isolate/Purify Protein
Insulin for
diabetics
Why Purify Proteins?
Pancreas
→
Identify Cells
→ Insert Plasmid into Cell
→
Isolate Gene
→ Cell Creates Insulin
→ Human Use
→
Insert Gene
→ Isolate/Purify Protein
Insulin for
diabetics
Why Purify Proteins?
Pancreas
→
Identify Cells
→ Insert Plasmid into Cell
→
Isolate Gene
→ Cell Creates Insulin
→ Human Use
→
Insert Gene
→ Isolate/Purify Protein
Insulin for
diabetics
Why Purify Proteins?
Pancreas
→
Identify Cells
→ Insert Plasmid into Cell
→
Isolate Gene
→ Cell Creates Insulin
→ Human Use
→
Insert Gene
→ Isolate/Purify Protein
Insulin for
diabetics
Why Purify Proteins?
Pancreas
→
Identify Cells
→ Insert Plasmid into Cell
→
Isolate Gene
→ Cell Creates Insulin
→ Human Use
→
Insert Gene
→ Isolate/Purify Protein
Insulin for
diabetics
Why Purify Proteins?
Pancreas
→
Identify Cells
→ Insert Plasmid into Cell
→
Isolate Gene
→ Cell Creates Insulin
→ Human Use
→
Insert Gene
→ Isolate/Purify Protein
Insulin for
diabetics
Why Purify Proteins?
Pancreas
→
Identify Cells
→ Insert Plasmid into Cell
→
Isolate Gene
→ Cell Creates Insulin
→ Human Use
→
Insert Gene
→ Isolate/Purify Protein
Insulin for
diabetics
How do you purify proteins?
Supernatant
Ni2+
Snap freeze on dry ice
Pellet
1. Lyse (cut)
open the cells.
2. Centrifuge to
create pellet.
3.Mix supernatant
with nickel beads.
How do you purify proteins?
1. Lyse (cut) open the cells.
2. Centrifuge to create pellet.
3. Mix supernatant with nickel beads.
4. Pass the supernatant through the column.
5. Add elution buffer.
6. End with a pure sample containing
only the fluorescent protein.
How do you purify proteins?
4. Pass the supernatant
through the column.
5. Add elution buffer.
6. End with a pure
sample containing
only the fluorescent
protein.
The “Central Dogma”
Transcription
Translation
DNA
RNA
Protein
Nucleic
Acids
Nucleic
Acids
Amino
Acids
Trait
Green Fluorescent Protein (GFP)
Discovery of GFP (1960’s)
Aequorea victoria
Osamu Shimomura
Co-winner of Nobel Prize
How does fluorescence work?
How does fluorescence work?
Excited state
Blue light
(High energy)
Green light
(Lower energy)
Ground state
Fluorescence vs. Bioluminescence
Natural Light
Scorpion- Natural Light
In the Dark
Scorpion- UV Light
Fluorescent organism:
Absorbs light at one wavelength (UV) and re-emits light
at a visible wavelength (color)
Bioluminescent organism:
Produces its own light.
Fluorescence vs. Bioluminescence
Fluorescent organism:
Absorbs light at one wavelength (UV) and re-emits light
at a visible wavelength (color)
Bioluminescent organism:
Produces its own light.