Purification of mFP from an overnight culture Laboratory 7

Why are proteins important in biology? Do Now Why are proteins important in biology? How are proteins used in the human body?

Take cells growing in broth: Objectives: Take cells growing in broth: Lyse (break open) From overnight LB/amp/ara culture Purify mFP from cell lysate using column chromatography

Introduction Once laboratories locate promising therapeutic protein: Then locate and isolate gene that encodes the protein Insert gene into plasmid (to clone gene) Cloning vectors Plasmid engineered to replicate in high numbers Within bacterial cell Expression vectors pARA-R with rfp gene Plasmid engineered specifically for protein expression Transformed cells Allowed to express protein Lysed to release synthesized protein from cell

Introduction Mutant fluorescent protein: 238 aa in size Fluorophore located in center Highly hydrophobic In order to purify (separate) protein: Look for differences in hydrophobicity Hydrophobic verse hydrophilic Some have regions that are both Hydrophobic regions will “hide” in interior of molecule How to isolate a single protein? E. coli we are using produces HIGH concentrations of mFP

Introduction Column chromatography Purification technique uses hydrophobicity to separate and purify proteins Plastic cylinder with resin Separating medium Contains small hydrophobic beads If mFP placed into solution of high salt concentrations: mFP molecule distorted Hydrophobic regions adhere to resin Hydrophilic proteins then continue down column and flushed away

Introduction mFP trapped in resin bed: Wash column with solution low salt concentration Will elute (wash out) moderately hydrophobic molecules with buffer Use solution of very low salt concentration to release mFP from resin beads

Materials Reagents: Equipment & Supplies: 2 mL LB/amp/ara culture E. coli Lysozyme (10mg/mL) Binding buffer, 4 M (NH4)2SO4 Column equilibration buffer, 2 M (NH4)2SO4 Elution buffer, 10mM TE 20% Ethanol 10% Bleach or other disinfectant TE (same as elution buffer) Equipment & Supplies: Centrifuge P-200 pipette and tips P-1000 pipette tips Chromatography column Microfuge tube rack 1.5 mL microfuge tubes Markers 6 mL waste collection tube Cell-contaminated waste bag Ring stand clamp

What will you need to do? Preparation day 1 – lysing the cells Preparation day 2 – mFP purification using column chromatography

What will you need to do? Chromatography columns Capped tightly Stopcocks closed Store upright to allow resin bed to form flat surface Use ethanol to rinse resin if splashed on sides Open stopcock and let ethanol drain from column Leave about 2mm layer above resin bed Set up on ring stand for model High enough for collection below Make sure resin bed visible When done: Flush columns with 4-5 mL elution buffer Flush columns with 3 mL 20% ethanol Cap tightly!!!!

Turn to page 7.4…….. Methods - today **Start with “preparing the mrfp sample” Page 7.4, step 7 Rest done ahead of time Columns will have equilibration buffer Dispense down to 1 cm above resin Turn to page 7.4……..

Methods - today Step 7 - For each column, we will use two tubes of lysed cells Step 9 – use ALL of the supernatant from both tubes, but do not touch pellet Step 10 – add EQUAL amount of “binding buffer” to what removed of supernatant from above Ex: if got 500 μL from step 9, add 500 μL of binding buffer Step 11 – Add ALL (from both tubes) of solution Step 11 – delete the phrase “using the same pipette you used to mix the solutions”

Methods Do not let the supernatant (red) run through the tube….once it is gone, it is gone!! Step 16 – as soon as the red hits the first level of the chromatography column tip, get the 1.5 mL tube ready…collect when the red drips!!

Purification of RFP from an overnight culture Bruce Wallace Overnight culture Cell pellet with RFP Lysed cells Pellet cell debris RFP with binding buffer

Mini-Essay practice Although this laboratory involved the expression of a sea anemone gene, cite some examples of human proteins that could potentially be expressed and purified using similar methods. What are the main aspects from this experience we need to be able to recall?

Closing: did we do these? Take cells growing in broth: Lyse (break open) From overnight LB/amp/ara culture Purify mFP from cell lysate using column chromatography