Download Laboratory Exercise #7: Column Chromatography of GFP proteins

Laboratory Exercise #7: Column Chromatography of GFP proteins
Introduction
The lab is designed so that you can isolate the GFP protein from your bacterial cells. The bacterial cells
you are working with have been genetically modified
– i.e. transformed – with a pGLO plasmid that
contains a gene for the Green Fluorescent Protein
(GFP) from the jellyfish Aequoria victoria. As the
bacterial cells express proteins from their
endogenous genome, they will also express the GFP
protein from the GFP gene within the pGLO plasmid.
You have observed this already. When bacterial cells
are grown in the presence of the sugar arabinose (to
activate the arabinose operon), GFP protein is
expression and excited with UV light. As a result,
your bacterial cells have glowed.
Because of their rapid duplication times and their
high levels of transcription and translation, bacteria
are used in the research lab for the production of
many proteins in large amounts. The problem
becomes how do you isolate your protein and eliminate contamination of your protein sample by the
endogenous proteins of the bacteria? A common method is the isolation of a protein using column
chromatography.
Chromatography is a powerful method used in the science field for separating and purifying desired
proteins from a sample. The purified proteins can then be used for many things, such as medicines to
treat human diseases or as enzymes that are part of household cleaning products. The chromatography
method described in this lab exercise is analogous to the processes used in industry to produce and
isolate large amounts of proteins for commercial value.
In order to isolate the GFP protein from your bacteria, you must first break open the cells. The easiest
way to do this is to freeze/thaw the bacterial cells. The physical breaking of the bacterial cell membranes
by freezing and thawing is made easier by mixing the bacteria with the enzyme known as lysozyme – a
general anti-bacterial enzyme. Lyzosome enzymatically cleaves sugars within the cell wall of the
bacterial. Freezing and thawing the bacteria after lysozyme treatment breaks the weakened cell walls and
the plasma membrane under it. This results in the lysis of the bacteria and the release of their internal
contents, forming what is called a lysate. One of the proteins in the lysate will be GFP.
In chromatography, a cylinder or column is filled with a “bed” of microscopic beads. The beads form a
“matrix” through which the proteins must pass before being collected. The matrix within the columns you
will be using has an affinity for the GFP protein but not for the other bacterial proteins. As the bacterial
lysate flows through the column, the GFP protein will adhere to the matrix and the other protein
contaminants will continue through the column.
Hydrophobic (water-hating) substances do not mix well with water. When they are mixed with salt
water, hydrophobic molecules will stick together. Proteins often contain numerous hydrophobic amino
acids. In salt water, these parts of the proteins will adhere to other hydrophobic substances. So a high salt
concentration will cause the 3D structure of a protein to change so that the hydrophobic regions of the
protein are exposed on the surface of the protein and the water-loving hydrophilic regions are more
shielded.
This chemical principle is what is being used in today’s lab on column chromatography. Specifically, the
kind of chromatography you will be performing is called hydrophobic interaction chromatography
(HIC) because the GFP protein will be purified using hydrophobic interactions with the matrix of the
column. To get the GFP to stick to the matrix beads, a highly salty Binding Buffer will be added to the
bacterial lysate to increase its salt concentration. The bacterial proteins are less hydrophobic will not
adhere to the matrix beads and will pass through the column. However, some of them may be
hydrophobic enough to weakly adhere to a bead. Because of this, the columns will be washed to ensure
the removal of any of these bacterial proteins. This wash will remove them but will leave the GFP protein
stuck to the beads. Once all impurities have been removed by washing, the removal of the GFP protein
from the bead can be accomplished by decreasing the salt concentration in the column. This is done by
adding an Elution Buffer to the column. The decrease in salt will change the 3D conformation of the GFP
protein once again – resulting in the exposure of the hydrophilic amino acids and the shielding of the
hydrophobic protein regions. This change in 3D shape decreases the affinity of the GFP protein for the
bead and increases its affinity for the surrounding buffer. As a result, the GFP lets go or “elutes” off of the
bead and can be collected from the column. You may see this elution if you expose the column to UV light.
What you might see is a fluorescent green “ring” passing down the column.
Required Materials for Lab
Common workstation:
Bacterial overnight culture from 32°C constant shaking
Lysozyme solution on ice
GFP chromatography columns
Equilibration Buffer (EqB)
Binding Buffer (BB)
Wash Buffer (WB)
Elution buffer (EB) = TE buffer
Collection tubes
Parafilm
Plastic transfer pipettes
1.5ml centrifuge tubes
Equipment needed:
Micropipettes and tips
UV lamps
Pre-lab activities
Record the purpose of this activity.
READ THE PROTOCOL. Be sure you understand what each step in this protocol is for.
Protocol
1. Obtain the following: one chromatography column, three collection tubes and aliquots of Equilibration
Buffer (EqB), Binding Buffer (BB), Wash Buffer (WB) and Elution Buffer/TE Buffer (TE). Label the 4
collection tubes, #1, #2, #3 and D
2. Also obtain your bacterial overnights that were grown at 37°C with shaking. Pipette 2.0 mL of the
LB amp and LB amp/ara overnights into labelled micro-centrifuge tubes. Pellet the bacteria by
spinning at maximum speed for 5 minutes.
3. Pour out the supernatants and observe the pellets under UV light and record your observations in your
notebook. Compare your pellets from LBamp cultures to LBamp/ara cultures.
THE FOLLOWING STEPS WILL BE PERFORMED USING THE LB AMP/ARA BACTERIAL PELLET.
4. Resuspend the LB amp/ara pellet in 250uL of TE buffer. Use a vortexer to completely resuspend the
bacterial cells
5. Using a plastic transfer pipet, add 1 drop of lysozyme solution to the resuspended bacterial pellets.
Mix the contents by gently inverting the tube a few times. Observe under UV light and record your
observations in your notebook.
6. Place the bacterial solution in the freezer until the solution freezes. Thaw the suspension in a water
bath set to 37C.
7. Centrifuge the tubes at maximum speed for 10 minutes to pellet the debris.
8. While your bacteria is spinning, prepare the chromatography column. “Flick” the GFP column like a
thermometer to collect the resin at the bottom of the column. Alternatively, you can tap the column
gently on the desk to settle the resin. Remove the top cap and snap off the bottom from the HIC column.
Place the column into a collection tube labelled #1 and allow the liquid to drain from the column (about 5
minutes). Completely discard this flow-through from the collection tube.
9. Place the drained column back into a collection tube #1. Add 1.0 mL of EqB to the top of the column
and allow to drain through. Repeat with another 1.0 mL of EqB. Completely discard the flow through.
This “medium” salt-solution prepares the matrix of the column so that it will bind protein.
10. Remove the bacteria from the centrifuge and immediately pipette 250uL of the supernatant into
the micro-centrifuge tube labelled BB. This tube contains 250uL of a high salt binding buffer and will
prepare your sample for loading onto the column.
11. Load 250uL of the prepared bacterial lysate onto the top of the column. Hold the pipet tip against
the side of the column wall, just above the surface of the matrix and let the supernatant drip down the
side of the column wall. Allow the bacterial lysate to completely drain through into collection tube #1.
This step binds the GFP protein to the column’s resin. Keep tube #1 and set aside.
12. After the column stops dripping, transfer the column to a new collection tube labelled #2. Wash the
column with 250uL of WB and allow to flow through. This step will wash away any impurities but will
not affect the GFP bound to the column. Keep tube #2 and set side.
13. After the column stops dripping, transfer the column to a new collection tube labelled #3. Elute the
GFP protein from the column with 750uL of EB/TE buffer and allow to flow through. Keep tube #3 and
set side.
14. Discard your GFP column in the trash. Dispose of collection tubes #1 and #2 and wash them
15. Compare your three collection tubes using UV light and record your observations in your notebook.
Parafilm collection tube #3 and
store at 4°C.