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 jellyfishAequoriavictoria. 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 yourbacterial 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 lightand 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 250uLof the supernatantinto themicro-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 750uLof 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.