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Protein PurificationProtein Purification
Protein PurificationProtein Purification
• What do you know about proteins?
• Why do we need to purify proteins?
• What are you curious about?
Bacteria now express cloned fluorescent protein…
Bacterial chromosome
Allow bacteria to grow for 1-3 days on plate with ampicillin.
Plasmid Uptake of foreign DNA, often a circular plasmid
Bacterial chromosome
What is Transformation?
Through transformation, bacteria now express cloned fluorescent protein.
Purified Fluorescent Proteins Purified Fluorescent Proteins Roger Tsien and Osamu ShimomuraRoger Tsien and Osamu Shimomura
Tsien
Osamu Shimomura
Purify a single protein of interest from over 4,000 naturally occurring E. coli gene products.
What is Protein Purification?
1. Lyse (cut) open the cells
Snap freeze on dry ice
Supernatant
Pellet
2. Centrifuge to create pellet
3.Mix supernatant with nickel beads
Purification Steps
4. Pass the supernatant through the column
5. Add elution buffer6. End with a pure
sample containing only the fluorescent protein
Column Chromatography
his-his-h
is-his-h
is-his
The “his tag”Fluorescent Protein with “his tag”
The His tag us how the protein attaches to the Nickel bead
Purpose of the Nickel Beads• Nickel bead binds
to His tag of FP (like two magnets)
• Now the Fluorescent Proteins are attached to the nickel beads and will not be able to flow through the column with the other proteins.
Ni2+
Separating FP from other proteins
The Nickel beads are too big to pass through the column, so the FP’s that are stuck to nickel beads are not able to flow through the cotton.
All other proteins will flow throughcotton ball into waste tube
Fluorescent Proteins
Elution
• FP are separated from nickel beads by the imidizole (elution buffer)
• Now that FP is no longer attached to the Ni bead, it can pass through the column
Ni2+
Imidazole
Histidine
Protocol
In our saliva, tears, spleen, lung, kidney
High concentration in chicken egg-white (our source of lysozyme).
Lysozyme was discovered accidentally in 1922 by Alexander Fleming by accident. Nasal drippings in the petri dish with bacterial culture, killing the bacterial cells.
LYSOZYMELYSOZYME
Viruses use lysozyme to break into the host bacterial cell allowing it to inject its DNA.
This technique involves freezing and then thawing the material.
Causes cells to swell and ultimately break as ice crystals form during the freezing process and then contract during thawing.
.
““Snap Freeze” Cell LysisSnap Freeze” Cell Lysis
his-his-h
is-his-h
is-his
The “his tag”Fluorescent Protein with “his tag”
The His tag us how the protein attaches to the Nickel bead
Purpose of the Nickel Beads• Nickel bead binds
to His tag of FP (like two magnets)
• Now the Fluorescent Proteins are attached to the nickel beads and will not be able to flow through the column with the other proteins.
Ni2+
Elution
• FP are separated from nickel beads by the imidizole (elution buffer)
• Now that FP is no longer attached to the Ni bead, it can pass through the column
Ni2+
Imidazole
Histidine
…to purified protein product
Fluorescent Protein PurificationFluorescent Protein Purification
From organism…
Central DogmaCentral Dogma of Molecular Biology of Molecular Biology
DNA---> mRNA---> Protein---> Trait
Transcription Translation
Fluorescent ProteinsFluorescent Proteins
Protein Structure – x-ray crystalography
Fluorescent ProteinsFluorescent Proteins
Green (GFP) Red (RFP)
In RESEARCH: Characterization of the function,
structure and interactions
Example: X-Ray Crystallography
In MEDICINE: Vaccines created from recombinant proteins
Example: Insulin
Why Purify Proteins?
Engineered Fluorescent Proteins
From GFP: From RFP: 1. Green 4. Cherry 2. Blue 5. Tangerine 3. Grape 6. Yellow
Protein PurificationProtein Purification
Now that you’ve purified your floursecent proteins – how else to you see that this process could be used?