Purification of GFP
By transforming E. coli with the pGLO plasmid, we introduced the GFP gene into these bacteria. We can identify transformed bacteria both by their ability to grow on media containing ampicillin and their altered phenotype (green fluorescence when exposed to UV light) when grown in the presence of arabinose. The altered phenotype, of course, tells us that the GFP gene is being expressed.
Often, simply observing a phenotypic change in transformed bacteria is not a sufficient endpoint for an experiment. In many cases, in fact, the introduced gene and its resulting protein will not result in an observable phenotypic change. For instance, consider the production of human insulin in bacteria. We cannot tell if bacteria are producing insulin simply by looking at the colonies. Moreover, we are not just interested in having the bacteria express human insulin; we want to isolate the insulin for medical uses. Thus, it usually is desirable to detect and purify the recombinant protein.
Many protein purification strategies can be used to purify different proteins. Proteins can be separated based on size, charge, or hydrophobicity, for example. Usually, the development of an efficient purification strategy for a specific protein is a long, labor-intensive process. In this experiment, we will use hydrophobicity chromatography to purify GFP. Then, we will determine the purity of our preparations by polyacrylamdine gel electrophoresis (PAGE).
pGLO transformed bacteria
Grow overnight cultures of pGLO transformed bacteria. One colony should be grown in nutrient broth + ampicillin. The other colony should be grown in nutrinet broth + ampicillin + arabinose. The bacteria should be grown at 37 degrees with moderate shaking.
1. Transfer approximately 1.5mL of each overnight culture to separate microfuge tubes. Centrifuge the tubes at 12,000 xg for 5 minutes to pellet the cells.
2. Discard the supernatant and observe the pellet under UV light.
3. Resuspend the pellets in 250uL of TE buffer. NOTE: TE (Tris-EDTA) is a mild buffer routinely used for many molecular biology protocols.
4. To each tube, add 1 drop of lysozyme. Lysozyme is an enzyme that destroys bacterial cell walls. It is found naturally in our tears and saliva as an initial defense against bacterial infections. Gently mix the contents of the tube.
5. Freeze your tubes at -70 degrees. This freezing and subsequent thawing will aide in lysing the cells.
6. After about 20 minutes, thaw the tubes and centrifuge at 12,000 xg for 10 minutes to pellet large cellular debris.
7. Remove the top and bottom from the hydrophobicity columns and allow the buffer to drain.
8. Add two 1 mL aliquots of equilibration buffer to the top of the column. Allow the buffer to drain to the 1 mL mark on the column, then cap the column until you are ready to proceed. The equilibration buffer raises the salt concentration of the column to match that of the cell lysate.
9. Transfer 250uL of your cell lysate supernatant to a clean microfuge tube and add 250uL of binding buffer to it. The binding buffer has a high salt concentration. The increased salt alters the conformation of the GFP protein such that it readily binds to the column matrix.
10. Uncap your column and place it in collection tube 1. Collect the buffer that drains out of the column and then add 250uL of lysate supernatant to the column matrix. Let it drain into collection tube 1.
11. Transfer the column to collection tube 2 and add 250uL of wash buffer to the column. Collect the liquid from the column in this collection tube. The wash buffer will aide in the removal of weakly bound proteins.
12. Add 750uL of TE buffer to the column and collect the eluent in collection tube 3.
13. Examine all three collection tubes under UV light, seal them with Parafilm, and store them in the refrigerator.
Through the use of size exclusion chromatography, we were able to fractionate based on size the proteins in a cellular extract. As we discussed in class, it is important after fractionation to assay the fractions in order to determine which fraction(s) contain your protein of interest. Usually, it also is useful to analyze your proteins via polyacrylamide gel electrophoresis (PAGE). This technique can provide at least two important pieces of information: the size of your protein and the relative purity of your preparation. If many additional proteins are in the same fraction as your protein of interest, then you do not have a very pure preparation. As you may guess, yield (how much of your protein you have) and purity often are inversely related. For the biochemist, developing a protein purification scheme that results in an optimal yield and purity can be challenging.
1. Transfer 10 ul of each of your fractions to
separate, clean microfuge tubes.
2. To each tube, add 10 ul of loading dye.
3. Cap tubes tightly and boil for 3 minutes.
4. Load approximately 20 ul of each sample into separate wells of a 12% polyacrylamide gel. NOTE: polyacrylamide is a neurotoxin. Wear gloves!
5. Also load 5 ul of a protein molecular weight marker into two wells.
6. Run the gel at 125 V for approximately 90 minutes.
7. Remove the gel from the plates and add enough Coomassie stain to cover the gel.
8. Gently shake for 45 minutes.
9. Remove (and save) the Coomassie stain and add destain to the gel
NOTE: Destain is 4.5 parts methanol, 4.5 parts water, and 1 part acetic acid.
10. Destain overnight and take a picture of the gel.
What size is GFP?
How pure is your preparation of GFP?