1. Purification of mFP from an overnight culture
Laboratory 7

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

3. 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

4. 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

5. 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

6. 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

7. 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

8. 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

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

10. 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!!!!

11. 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……..

12. 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”

13. 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!!

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

15. 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?

16. 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