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?