Protein 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 Roger 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 buffer 6. End with a pure sample containing only the fluorescent protein Column Chromatography

his-his-his-his-his-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. Ni 2+

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 through cotton 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 Ni 2+ 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. LYSOZYME 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 Lysis

his-his-his-his-his-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. Ni 2+

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 Ni 2+ Imidazole Histidine

…to purified protein product Fluorescent Protein Purification From organism…

Central Dogma of Molecular Biology DNA---> mRNA---> Protein---> Trait TranscriptionTranslation

Fluorescent Proteins

Protein Structure – x-ray crystalography

Fluorescent 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 Purification Now that you’ve purified your floursecent proteins – how else to you see that this process could be used?