The Expression and Purification of Recombinant Green Fluorescent Protein (rGFP) From E. coli strain, BL21(DE3), Using Ni2+-Agarose Affinity Chromatography

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Abstract:
The purpose of these series of experiments was to express and purify recombinant Green Fluorescent Protein (rGFP) from the E. coli strain, BL21(DE3) by beginning with its purification via a Ni2+-agarose affinity chromatography column. The His6 tag of the rGFP bound to the Ni2+-agarose column and washes and elutions were obtained, with elution 3 containing the most amount of fluorescence at approximately 12,000 RFUs.

From here, a Bradford assay was performed in order to determine how much protein was in each sample and an SDS-PAGE/Coomassie Blue analysis was done to determine the size and purity of rGFP in the elution 3 sample. The sample came out to be about 34 kDa and was about 75% pure. Lastly, a Western Blot was performed with binding of primary and secondary antibodies to prove that the protein of interest, rGFP, had indeed been expressed and purified. Introduction:

Osama Shimomura first discovered Green Fluorescent Protein (GFP) in 1962. The discovery was made inside the bioluminescent jellyfish A. equorea victoria. GFP was found to be a protein of 238 amino acids and had a molecular weight of 26,888 kDa. He was later able to characterize the structure of one of GFP’s unique features- its chromophore, which causes the protein to fluoresce. Later in 1985, Douglas Prasher became the first person to clone GFP (Prasher, 230).

For these experiments, a recombinant form of GFP that contained a Histidine6 tag (His6 tag) on the N-terminus of the protein, as well as an X-press epitope tag that begins at the 24th amino acid residue was taken. The His6 tag’s metal ion binding feature was critical during the Ni2+-agarose affinity chromatography because it was able to bind to the nickel inside the column and thus allowed rGFP to be purified since other proteins would be washed out.

The purpose of these series of experiments was to allow the expression and purification of rGFP so that further analysis could be done, such as howto determine the purity, size, and total yield of a protein. Finding out this information would give a better understanding of the characteristics of GFP and its significant properties that could lead to further scientific and medical discoveries.

Page 2 Expression and Purification of rGFP from E. coli Essay

Materials and Methods: (Referenced from the “Biochemistry Lab Manual”) Expression ofrGFP in E. coli- Using a single bacterial colony of strain G (BL21pLysS, pRSETA-GFPuv strain from E. coli), 10 ml of LB media [100ug/ml Amp; 25ug/ml Cam] was inoculated and was grown overnight at 37°C with vigorous shaking until the culture was saturated. Then 500 ml of liquid LB growth media [100ug/ml Amp; 25ug/ml Cam in 1 liter baffled flask, pre-warmed-30°C] was inoculated with ~4ml of the saturated overnight culture so the culture had an OD600 of 0.1. This culture was grown overnight at 37°C with vigorous shaking until OD600 of 0.5 was reached. A bacterial pellet designated “G0” was obtained at time equals zero by pelleting 1ml of culture and discarding the supernatant.

The culture was then induced with IPTG (1mM final concentration) and allowed to grow. After 3 hours, a “G3” bacterial pellet was obtained and both G0 and G3 were stored at -20°C. Also, 15 ml of the culture was pelleted and the supernatant was discarded, leaving a “G3-15ml” pellet and was stored at -20°C to be used later as the starting material to isolate rGFP. Purification of rGFP using Ni2+-Agarose Affinity Chromatography- The rGFP crude extract was prepared before performing the Ni2+-agarose affinity chromatography.

The freeze-thaw method was used on the G3-15ml bacterial pellet. The slow freezing at -20°C causes small ice crystals to form within the bacteria, damaging the bacterial membrane. Addition of 500ul of breaking buffer [10mM Tris, pH 8; 150mM NaCl] twice to the G3-15ml pellet and immediately pipetting the breaking buffer up and down causes the pellet to thaw and the cytoplasm of the bacteria to be released, creating a homogeneous solution. After vortex for 5 minutes, the solution was placed in a 37°C water bath for 10 minutes. It was then transferred to a rotating platform shaker in a dry air 37°C incubator for 20 minutes. Once the bacteria had lysed, it was centrifuged at 14,000xg, 4°C, for 10 minutes.

The supernatant was decanted into a new tube and labeled, GCE (rGFP crude extract). The Ni2+-Agarose column was made bypacking in a small amount of glass wool inside a 3 ml syringe. Once secured onto a ring stand with a clamp, a luer-lock was fastened onto the end of the syringe. 2ml of breaking buffer was added to the column and a few drops were excreted before closing the luer-lock to prevent air bubbles.

With at ~500ul of breaking buffer in the column, 1ml of 50% Ni2+-agarose slurry was added to the column and the luer-lock was opened to pack the agarose matrix, creating a bed volume of 500ul. The column was then washed with breaking buffer in order to take out any ethanol that the agarose had been stored in. Next, 1ml of GCE was applied and given 5-10 minutes for the his6 tag on the rGFP to bind with the Ni2+-agarose matrix. The luer-lock was then opened to allow ~0.5ml of the effluent to flow into a tube and labeled, W1.

The next 0.5ml of the effluent that came out was labeled, W2. Then, breaking buffer was pipetted into the column in 0.5ml increments and each was collected in separate tubes and labeled W3-W10. After the column was rinsed with 5ml of breaking buffer, an elution buffer [10mM Tris, pH 8.0; 150mM NaCl, 300mM imidazole] was added in 0.5ml increments and each of these were collected and labeled E1-310. Determining Protein Concentration of rGFP Fractions- A Bradford assay was used to determine the total protein amount in washes 1-6 and elutions 1-6. First a Bradford standard curve was created using 6 different amounts of Bovine Serum Albumin (BSA)- 0ug, 2.5ug, 5ug, 10ug, 15ug, and 20ug.

Each sample was made from a 1mg/ml BSA stock solution and mixed with enough water to bring the volume to a total of 50ul in each tube. 1ml of Bradford reagent was added to each and mixed by vortexing and then incubated at room temperature for 10 minutes. Using a microplate reader, the absorbance at 595nm of each sample could be found. By plotting the data [Absorbance (595nm) vs BSA (ug)], the standard curve was created. The same procedure was done for the W1-6 and E1-6 samples by taking 50ul of each sample and adding 1 ml of the Bradford reagent.

Once absorbance was measured, extrapolation of these absorbance values on the standard curve could be done in order to determine the amount of total protein that was present in each sample. SDS-PAGE/Coomassie Blue Analysis- The SDS-PAGE gel was created with 12% resolving gel (used 4X stacking buffer) and 5% stacking gel (used 4X resolving buffer). Both gels contained water, 30% Acrylamide, 10% APS, and TEMED. Using a device made of 2 pieces of glass, the resolving gel was poured in and was solidified before adding the stacking gel on top. Once it polymerized, a comb was inserted to create the wells in which the samples would be placed. This device was then put inside an electrophoresis tank, which was then filled with electrophoresis buffer. Once the samples (GO, G3, GCE, W3, W4, E3, E4) were prepared with 4X sample loading buffer, they were placed inside the wells and electrophored for 45 minutes at 200 volts.

The gel was then removed, stained with Coomassie Blue, and de-stained so that the size and purity of each sample containing the rGFP protein could be determined. SDS-PAGE/Western Blot Development- The SDS-PAGE gel was created, the same prepared samples were loaded, and the electrophoresis was run the same way. This time, the gel was placed inside a cassette (transfer apparatus) with filter paper being placed at the base, then the nitrocellulose (NC) membrane, then the gel, and more filter paper before placing the lid on top.

Once the proteins migrated onto the NC membrane, it was stained with 20ml of Ponceau S and de-stained in order to determine the efficiency of the transfer, the orientation of the blot, and to mark the ladder. The blocking step was performed by adding 30ml of 5% non-fat dry milk/TBS solution and incubated for 30 minutes. The membrane was then washed by adding of 30ml of 0.05% Tween 20/TBS and incubated (3X). It was then probed with 7ml of mouse IgG anti-Xpress epitope MAb solution and incubated. The same washing step was repeated 3 times before probing the membrane with 7ml of Sheep IgG anti-mouse IgG conjugated horseradish peroxidase polyclonal anti-serum solution and incubated. After a final wash, 7ml of TMB substrate solution was added, and the membrane was incubated until the desired color intensity was achieved. Results:

The expression of rGFP undergoes a process that begins with the induction of IPTG. If no IPTG is induced, the lac repressor will inhibit expression of T7 RNA Polymerase. IPTG inhibits this lac repressor so that a large enough amount of T7 RNA Polymerase is made to overwhelm the inhibition by lysozymes. In figure 1 it is seen that T7 RNA Polymerase allows the T7 promoter in the plasmid to transcribe and translate the rGFP gene. Ampicillin is also to the plasmid to maintain selectivity and protect it from contamination so that only rGFP is being expressed. The T7 promoter will allow the T7 RNA Polymerase to bind to the His6-Xpress-GFPuv tag that’s next to the T7 Promoter and then expression of rGFP will begin. At time
equals zero, our G0 sample was collected and then 3 hours after induction with IPTG, our G3 was collected. Both samples were measured qualitatively (handheld UV lamp) and quantitatively (spectrofluorometer).

Looking at figure 2, a closer look at the rGFP gene was analyzed. The results showed that the entire gene was 279 amino acids long and contained the His6 tag near the N-terminus, the Xpress epitope tag between the His6 tage and the rGFPuv, and finally the rGFPuv near the C-terminal. Inside the rGFPuv, is the chromophore structure that causes the fluorescence. Since the molecular weight of a single amino acid is 120 Daltons, the MW of this protein can be calculated by multiplying its number of amino acids (279) by the MW of each amino acid (120) giving 33,480 Daltons.

A combined activity/elution profile for rGFP was made in order to compare the amount of fluorescence with the total amount of protein in each sample (W1-6, and E1-6) (figure 3). The graph showed the washes to have larger amounts of protein, as was expected since all the undesired proteins were being rinsed out. And although the elutions fewer amounts of protein, they had a greater amount of RFU’s, which indicated being on the right track to knowing whether rGFP had been purified.

In the next experiment, an SDS-PAGE gel was created and stained with Coomassie Blue (figure 4). Between elutions 2 and 3, the third one looked the most prominent and therefore was assumed to be the rGFP band. It was located around the 34 kDa marker on the ladder, which is fairly close to the calculated MW of the rGFP protein. Other bands smaller than this were also visible, but at this time could not be identified as a contaminant or rGFP with C-terminus degradation. The band looked to be about 75% pure.

In the final experiment, a Western Blot was developed in order to determine whether the proteins had transferred onto the NC membrane and if it was indeed the rGFP protein (figure 5). Undesired proteins were blocked with 5% non-fat dry milk/TBS solution, and then the proteins left were probed with the primary antibody. This primary antibody should have bound to the Xpress-epitope tag in rGFP and when probed with the secondary antibody, it should have bound on several spots of the primary in order to amplify the signal. After washed with TBS substrate solution, the Western Blot proved that rGFP had been successfully purified and that it was the band at 34 kDa. The Western Blot also showed that the smaller bands were merely contaminants since they did not show up in the final product.

Conclusion/Discussion:
The attempt to express and purify rGFP from these series of experiments was successful. The Ni2+-Agarose affinity chromatography purified the protein by allowing other proteins to be washed out and rGFP to be collected in the elution samples. The qualitative (handheld UV lamp) and quantitative (spectrofluorometer) measurements taken showed some fluorescence in the washes and this was probably due to the N-terminus (containing the His6 tag) being degraded and therefore not being able to bind to the column.

The third elution (E3) ended up having the most amount of rGFP and this was proven with the Bradford assay. The SDS-PAGE/Coomassie Blue Analysis experiment then helped to identify the purity and size of the rGFP in this third elution. With the purity being approximately 75% and the band showing up around the 34 kDa marker, the conclusion was made that rGFP was indeed purified by the Ni2+-Agarose column and that the calculated molecular weight was similar to what was shown on the gel. However, there were some bands at a lower molecular weight that did show up on the gel, but at this time it could not be determined whether they were contaminants or rGFP that had had their C-terminus degraded.

In order to determine that the band found in E3 was in fact rGFP, the SDS-PAGE process was done again and this time stained with Ponceau S and a Western Blot was performed. The primary antibody was able to bind to rGFP’s unique feature, the Xpress-epitope tag, and then the secondary antibody amplified and conjugated it. Because the antibodies were able to bind and the visible markings showed up in the Western Blot, this was able to prove that the bands on the gel were indeed rGFP. The fact that they didn’t bind to the lower bands with the smaller molecular weight brought on the conclusion that there were simply contaminants that did not contain the Xpress-epitope tag.

After these conclusions were made, follow-up experiments could be performed to see how useful GFP could be. One experiment that could be done is taking the gene that encodes this green fluorescent protein and transfecting the cells of another organism to see if it could produce fluorescence as well. Another experiment could be taking GFP and using it as a marker to tag a protein of interest to see this protein’s activity and its’ localization in the cell (Chalfie, et. al.). Experiments with GFP could prove to be very helpful in the world of science by discovering the expression and activity of proteins and how they can affect living organisms.

References:
Rippel, Scott. Biochemistry Lab Manual. Print.
Prasher, Douglas C., Virginia K. Echenrode, William W. Ward, Frank G. Prendergast, and Milton J. Cormier. “Primary Structure of the Aequorea Victoria Green-Fluorescent Protein.” Gene 111 (1992): 229-233 Chalfie, Martin. “Green Fluorescent Protein as a Marker for Gene Expression.” National Center for Biotechnology Information. U.S. National Library of Medicine. Web. 04 Aug. 2013. http://www.ncbi.nlm.nih.gov/pubmed/8303295.

Figure 5. Western Blot of rGFP Fractions.

Figure Captions

Figure 1. Plasmid Map of rGFP from E. coli strain, BL21(DE3). IPTG inhibits the lac repressor protein allowing T7 RNA polymerase to be produced. The PT7 promotes expression on His6-Xpress-GFPuv and placed in a media that contain ampicillin to maintain selectivity so that only rGFP is produced

Figure 2. The schematic diagram of rGFP from the E. coli strain, BL21(DE3). The numbers indicate where each amino acid sequence begins. The Histidine-6 tag begins at the 5th amino acid residue. The Xpress Epitope tag begins at the 24th amino acid residue. Finally, the rGFPuv sequence, being 238 residues long, begins at the 39th amino acid residue. It also contains the chromophore (65-67 residues of rGFPuv, Ser-Gly-Tyr) which is located from the 103rd-105th residue. This diagram displays the entire sequebce of the
rGFP protein, coming out to be 279 amino acids long.

Figure 3. Combined activity/elution Profile of rGFP during washes 1-6 and elutions 1-6 in a Ni2+-agarose column. The graph shows RFU’s (left) and total protein in ug (right) for each wash and elution. Once the rGFP had been purified in the Ni2+-agarose column, a breaking buffer (10mM Tris, pH 8.0; 150mM NaCl) was used to create the washes by pipetting the buffer in 0.5ml increments and collecting each in separate tubes labeling them W1-W6. Then, an elution buffer (10mM Tris, pH 8.0; 150mM NaCl; 300mM imidazole) was collected in increments of 0.5ml and labeled E1-E6. We could measure the amount of RFU’s each sample had using 200ul of each fraction and placing them in a spectrofluorometer. E3 showed to have the greatest amount of fluorescence. Using the Bradford assay, the total protein of each sample was determined by extrapolating points from the standard BSA curve. Both values were scaled up.

Figure 4. SDS-PAGE gel/Coomassie Blue of rGFP fractions from E. coli strain, BL21(DE3). The gel was created by using 12% resolving gel which was composed of water, 4X resolving buffer (0.75M Tris, pH 8.8, 0.4% SDS), 30% Acrylamide (29.2% w/v acrylamide, 0.8% w/v bis acrylamide), 10% APS (ammonium persulfate), and TEMED (tetramethylenediamineAsds). Once solidified, a 5% stacking gel composed of water, 4X stacking buffer (0.25M Tris, pH 6.8, 0.4% SDS), the same 30% Acrylamide, 10% APS, and TEMED was added. Once the stacking gel was polymerized, the gel was ready to be loaded with fractions of the E. coli strain, BL21(DE3), that we collected from previous lab. A molecular weight marker (ladder) was also loaded onto the gel. Going left to right, lanes 1and 2 are disregarded, the G3, G0, GCE, W1, W2, E2, E3, ladder. The gel was placed in a gel electrophoresis tank and filled with electrophoresis buffer (450ml H2O, 50ml 10X stock solution (30g Tris, 144g glycine, 10g SDSD per liter).

Figure 5. Western blot of the rGFP samples. An SDS-PAGE gel was created with 12% resolving gel and 5% stacking gel and ran through the electrophoresis procedure. The gel was then placed in a cassette and on top of a nitrocellulose membrane so that the protein could transfer from the gel to
the membrane. The Ponceau S staining identified the presence of rGFP. It was then blocked with 5% non-fat dry milk/TBS solution and probed with a primary antibody, mouse IgG anti-Xpress epitope MAb solution. It binds uniquely to the Xpress-epitope tag in rGFP so it can be isolate. It was then probed with the second antibody, Sheep IgG anti-mouse IgG conjugated horse radish peroxidase polyclonal anti-serum solution. This would amplify and conjugate the rGFP and finally, it was washed with TMB substrate solution to create the color intensity desired.

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