protein purification with fluorescent proteins. laboratory introduction what is a protocol?what is a...
TRANSCRIPT
Protein Purification
with fluorescent proteins
Laboratory Introduction
What is a protocol? What is a protein?
Why do scientists use protocols?
Why would we need topurify proteins?
Now let’spractice
pipetting!
Organisms produce thousands of different proteins, each having a different function…
HormonesHair and Nails
Structural Support
Enzymes
Muscle Contraction
Antibodies
Receptors, membrane channels
Nutrient Storage
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)
Protein Structure
Protein Structure
1° = amino acids
2° = basic structure(hydrogen bonds)
3° = 3D structure
4° = interactionof subunits
Protein Structure
Structure determines
function.
Now you be the protein!
The “Central Dogma”
DNA RNA ProteinTranscription Translation
NucleicAcids
NucleicAcids
AminoAcids
Trait
The “Central Dogma”
DNA mRNA Protein Trait
Making a Protein in the Lab
E. coli bacterial cell
Bacterial genome
Plasmid
We use E. coli bacteria “transformed”with a plasmid (a loop of DNA).
“Transformation”
Making a Protein in the Lab
E. coli bacterial cell
The bacteria now express (make)the fluorescent protein.
DNA RNA Protein
…to purified protein product
From organism…
Engineered Fluorescent Proteins
Engineered Fluorescent Proteins
From GFP: From RFP: 1. Green 4. Cherry 2. Blue 5. Tangerine 3. Grape 6. Yellow
We have six different plasmids!
Our Plasmids
AmpR
Ampicillinresistance gene
FP gene
Plasmid Mix 1
GFP
BFP
Grape
Plasmid Mix 2
YFP
Tangerine
Cherry
“Ampicillin resistance” means the bacteria will
survive even when exposed to antibiotics!
Why Purify Proteins?
Research
• to make vaccines • to treat disorders
• to understand structure
Medicine
Pancreas → Identify Cells → Isolate Gene → Insert Gene
→ Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein
→ Human Use
Why Purify Proteins?
Insulin for diabetics
Pancreas → Identify Cells → Isolate Gene → Insert Gene
→ Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein
→ Human Use
Why Purify Proteins?
Insulin for diabetics
Pancreas → Identify Cells → Isolate Gene → Insert Gene
→ Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein
→ Human Use
Why Purify Proteins?
Insulin for diabetics
Pancreas → Identify Cells → Isolate Gene → Insert Gene
→ Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein
→ Human Use
Why Purify Proteins?
Insulin for diabetics
Pancreas → Identify Cells → Isolate Gene → Insert Gene
→ Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein
→ Human Use
Why Purify Proteins?
Insulin for diabetics
Pancreas → Identify Cells → Isolate Gene → Insert Gene
→ Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein
→ Human Use
Why Purify Proteins?
Insulin for diabetics
Pancreas → Identify Cells → Isolate Gene → Insert Gene
→ Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein
→ Human Use
Why Purify Proteins?
Insulin for diabetics
Pancreas → Identify Cells → Isolate Gene → Insert Gene
→ Insert Plasmid into Cell → Cell Creates Insulin → Isolate/Purify Protein
→ Human Use
Why Purify Proteins?
Insulin for diabetics
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
5. Retrieve the protein of interest
3. Distinguish the protein of interestNi2+
4. Separate the protein of interest
“Column Chromatography”
1. Lyse (cut) open the cells.
Snap freeze on dry ice
Supernatant
Pellet
2. Centrifuge to create pellet.
3.Mix supernatant with nickel beads.
How do you purify proteins?
Ni2+
Freezing and then thawing…
…causes ice crystals to break the cell open.
“Snap Freeze” Cell Lysis
How do you purify proteins?
Lysozyme is a naturally occurring enzymethat is used to break open cells.
Lysozyme
his-his-h
is-his-h
is-his
The “his tag”Fluorescent Protein with “his tag”
The “his tag” is how the protein attaches to the “nickel bead”!
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!
Purpose of the 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?
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.
Fluorescent Proteins
How do you purify proteins?
FP are separated from nickel beads by the imidazole (elution buffer).
Now FP is small enough to pass through the column.
Ni2+
Imidazole
Histidine
How do you purify proteins?
Elution
Purify a specific protein from over 4,000 naturally occurring E. coli gene products.
How do you purify proteins?
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.
Green Fluorescent Protein (GFP)
Osamu ShimomuraCo-winner of Nobel Prize
Aequorea victoria
Discovery of GFP (1960’s)
Jellyfish
Amphipod Spider’s palps
Fluorescent Organisms
Corals
How does fluorescence work?
How does fluorescence work?
Green light(Lower energy)
Excited state
Ground state
Blue light(High energy)
Fluorescence vs. Bioluminescence
Scorpion- UV Light
Scorpion- Natural Light
Fluorescent organism:Absorbs light at one wave-
length (UV) and re-emits light at a visible wavelength (color)
Bioluminescent organism:
Produces its own light.
Natural Light
In the Dark
Fluorescence vs. Bioluminescence
Fluorescent organism:Absorbs light at one wave-
length (UV) and re-emits light at a visible wavelength (color)
Bioluminescent organism:
Produces its own light.
GFP
RFP
Roger Tsien and Rainbow Proteins
Roger Tsien and Rainbow Proteins
Protocol Summary
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.
Ni2+