Abstract:
Green fluorescent protein (GFP) is a bioluminescent protein that is found in Aequorea Victoria, a species of Jelly fish. The goal of the experiment was to express and purify a recombinant form of GFP (rGFP) using Ni2+ agarose affinity chromatography technology. The His6/Xpress epitope tagged rGFP gene’s overexpression occurred in the BL21 (DE3) <pLysS> <pRSETA-GFPUV> E. coli strain. Following a Bradford assay and spectrofluorometric analyses, the fluorescent properties and protein amount of each of the wash and elution fractions obtained from the column were determined. The highest fluorescent activity of approximately of 88,000 and 85,000 relative fluorescent units (RFUs) were in elution fractions 2 and 3 (E2 and E3). Based on the standard curve of the Bradford assay, the protein amount of E3 was 30 µg. The specific activity of E3 was then calculated to be approximately 245,600 RFU/mg total protein. From the results from the SDS PAGE analysis, the purity of rGFP in the E3 lane was estimated to be 35%, so the yield of rGFP was calculated to be about 11 µg. Although its calculated molecular weight is approximately 32 kD, the relative and estimated molecular weights of rGFP were determined to be approximately 34 kD and 36 kD, respectively. Lastly, the presence of rGFP was verified using a western blot analysis.
Introduction:
In 1962, Osamu Shimomura reported his discovery of Green Fluorescent Protein, which is more commonly referred to as GFP, in Aequorea victoria (Pan, et. al., 2018). GFP is the protein responsible for giving Aequorea victoria, a class of jelly fish, to appear green to the human eye. The information Shimomura obtained led to the discovery of GFP’s protein sequence. GFP is composed of 238 amino acids that combine to create an approximately 27 kD protein (Pan, et. al., 2018). The GFP protein is extremely stable, meaning even when environmental conditions are changed its protein structure remains intact (Pan, et. al., 2018). The structure of the GFP protein consists of a chromophore that is surrounded, and, thus, protected by, a barrel with 11 strands (Pan, et. al., 2018). The chromophore present within GFP is responsible for the fluorescence of GFP, because once light hits the chromophore at 395 nm the light is emitted at 510 nm, which is what humans see as green (Pan, et. al., 2018).
In our experiment, a recombinant form of the GFP protein sequence was used. The pRSETA-GFP sequence that is expressed in the bacteria of our experiment contains an additional His6 tag. The His6 tag contains 6 consecutive residues and is fused to the N-terminus of the pRSETA-GFP sequence (Pan, et. al., 2018). rGFP can be purified using nickle (Ni2+) agarose affinity chromatography. The His6 tag readily binds to the agarose column, which allows for the pRESTA-GFP molecules to be isolated from a mixture of proteins. Once the rGFP is bound to the column, high concentrations of imidazole can be used to release, or elute, the rGFP off the column. Subsequent Bradford assay and SDS PAGE technology can be used to confirm that the protein obtained from the column is the desired rGFP.
The purpose of our experiment was to express a recombinant form of rGFP in a BL21 (DE3) <pLysS> <pRSETA-GFPUV> E. coli strain and purify this rGFP utilizing Ni2+ affinity chromatography. The data obtained from the Bradford assay, SDS PAGE, and western blot analyses were combined to determine the total protein amount, yield, specific activity, relative molecular weight, and detect the presence of rGFP.
Materials and Methods:
Expression of rGFP in E. coli
10 ml of LB growth medium containing 25µg/ml Cam and 100µg/ml Amp was inoculated with a single colony of BL21<DE3>pLysS, pRSETA-GFPUV (G strain). The 10 ml sample was grown overnight at 37ºC while being vigorously shaken until the culture was saturated (cloudy). Approximately 4 ml of the overnight saturated solution was used to inoculate 500 ml of LB growth medium. This 500ml growth medium was grown at 37ºC and vigorously shaken until its OD600 value reached 0.5. Once the culture reached an absorbance of ~0.5, 1 ml of the culture was pelleted into a 1.5 ml centrifuge tube labeled G0, which was then stored at -20ºC and will be analyzed further using SDS page gel. The remaining culture was induced with 1mM IPTG and was incubated and allowed to grow for an additional 3 hours. After the 3 hour incubation period, 1 ml of the culture was pelleted into a 1.5 ml centrifuge tube labelled G3, with its supernatant discarded, ad stored at -20ºC for subsequent analysis using SDS page gel. Also, 15ml of the culture was pelleted into a 15 ml centrifuge tube and was labeled G3-15ml and stored at -20ºC to be used to later isolate rGFP.
Preparation of rGFP crude extract
1ml of breaking buffer, which is composed of 10mM Tris, and 150mM NaCl, was transferred into the G3-15ml tube. Immediately after the addition of breaking buffer, the solution was continuously pipetted up and down until the pellet appeared to be completely dissolved. After being pipetted into a 1.5 ml centrifuge tube, the dissolved solution was vortexed for 5 minutes. The mixture was incubated in a 37ºC water bath for 10 minutes and then the tube was placed in a platform shaker for about 20 minutes to be incubated at 37ºC in dry air. The mixture was afterwards centrifuged at 14,000xg at 4ºC for 10 minutes. A qualitative analysis of the mixture was conducted using a UV lamp. The pellet was discarded, but the supernatant was decanted into a centrifuge labeled GCE.
Preparation of the Ni2+-Agarose column
A 3ml syringe containing ~ 0.25ml of glass wool was attached to a ring stand as vertically as possible with the output end facing down. After 1ml of buffer was added to the syringe, a closed luer lock that was full of buffer, to eliminate air bubbles, was added to the bottom of the syringe. An additional 2ml of breaking buffer added into the syringe. The luer lock was then opened to allow for dripping to occur, but then closed again. With the luer lock now closed, at least 500µl of breaking buffer was added to the syringe and 0.5ml bed volume of Ni2+ was added to create the column. To ensure the column was packed with agarose, the luer lock was opened again. An additional 5ml of breaking buffer was added to wash any remaining ethanol from the column and then the luer lock was closed again.
Washing and Elution of rGFP onto the Column
The GCE was brought to the desired 1ml volume by adding breaking buffer. The GCE was then added to the column, which is when the luer lock was opened. The 1.5ml centrifuge tube labeled sampled (W1) was the first 0.5ml collected from the syringe. W2 was the 1.5ml centrifuge tube that replaced W1 to collect the drainage from the column until the remaining 0.5ml had drained from the column. Subsequently, 0.5ml of breaking buffer was added to the to the column and collected bellow until another 8 washed had been collected in centrifuge tubes W3-W10. The column was then washed with 5ml of breaking buffer, which was not collected. The elution process was conducted by adding the elution buffer (10mM Tris, 150mM NaCl, and 300mM imidazole) for a total of 10 0.5ml elutions in centrifuge tubes labeled E1-E10. Centrifuge tubes W1-W6 and E1-E6 were visualized under UV lamp for qualitative analysis. Lastly, a spectrofluorometer was used to quantify the fluorescence of samples W1-W6 and E1-E6.
Bradford Assay Method for Determining Total Protein Concentration
Perform Bradford assay on 6 different known Bovine Serum Albumin (BSA) protein amounts, 0 µg, 2 µg, 4 µg, 6 µg, 8 µg, and 10 µg, to generate a standard curve. In a microtiter dish, add water, 50 µl of BSA, and 200 µl of Bradford assay, in that order, to obtain a final assay volume of 250 µl. After a 10-minute incubation period at room temperature, measure the absorbance of the samples at 595 nm. Once the standard curve is generated, perform triplicate Bradford assays on the W1-W6 and E1-E6 samples, using 50 µl aliquots of the protein samples in place of the BSA. However, perform the Bradford assays in singlicate for the samples first. If the absorbance values are outside of the standard curve, adjust the volume of the protein sample to ensure that the value will fall within the standard curve’s absorbance values for the subsequent two assays. Determine the total protein amounts in the W1-W6 and E1-E6 samples using the standard curve.
Using SDS-PAGE/Coomassie Blue Analysis to Determine Protein Purity
After cleaning the thick and thin plates with water and ethanol and assembling the plate clamp apparatus, pour the 12% resolving buffer (0.75M Tris pH 8.8, 0.4% SDS). Wait approximately 30 minutes to allow the resolving buffer to polymerize, then pour the 5% stacking buffer (0.25M Tris pH 6.8, 0.4% SDS) and immediately insert the comb. During the 30-minute polymerization waiting period for the stacking buffer, prepare the G0, G3, GCE, and the two wash and two elution fractions with the highest fluorescence in 1.5 ml centrifuge tube. Once the stacking buffer is polymerized, transfer the gel into the electrophoresis tank and load the samples and molecular weight ladder on the gel. Run the gel at 200V for approximately 45 minutes. Remove the gel. Stain and then destain the gel with Coomassie Blue dye.
Using Western Blot to determine Presence of rGFP
Perform an electrophoresis procedure as the one above. Transfer the gel into a western blot sandwich. Place the nitrocellulose membrane into a tupperware and stain with Ponceau S dye. After incubating with the dye for 2 minutes, discard the stain and mark the molecular weight ladder. Then, incubate the membrane with blocking solution (5% non-fat dry milk/TBS/0.05% Tween-20) for 30 minutes. After discarding the blocking solution, probe the membrane with primary antibody by adding the mouse anti-Xpress epitope MAb solution and incubating the membrane for 45 minutes. Discard the primary antibody solution and wash and incubate the membrane with TBS/0.05% Tween 20 for 5 minutes. Once the wash solution is discarded, probe the membrane with secondary antibody by adding the Sheep anti-mouse IgG conjugated horse radish peroxidase polyclonal anti-serum solution and incubating the membrane for 45 minutes. Discard the secondary antibody and perform the same washing procedure as described above using TBS/0.05% Tween 20 again. Perform a final washing step using TBS instead of TBS/0.05% Tween 20. After discarding the TBS from the final wash procedure, add TMB to the membrane and incubate. Once the desired color intensity is attained, transfer the membrane into a tupperware full of water to halt the development process. Dry the nitrocellulose membrane and use a camera to record the results as quickly as possible.
Results:
An IPTG-induced T7 RNA polymerase expression system was utilized to control the expression of rGFP in the BL21 (DE3) E. coli strain. After being placed in LB growth media, ane single bacteria stain G was treated with the <pLysS> and <pRSETA-GFP> genes. The pRSETA-GFP gene plasmid map can be seen in Figure 1. The T7 promoter on the pRSETA-GFP plasmid regulates the expression of the rGFP sequence. The T7 promoter itself is controlled by T7 RNA polymerase. In the presence of T7 RNA polymerase, the T7 promotor is activated, which leads to the expression of rGFP in the cells. However, if T7 RNA polymerase is not present, the T7 RNA promotor is not activated, so rGFP is not expressed. T7 RNA polymerase expression is inhibited by the Lac repressor protein produced by E. coli. The few T7 RNA polymerase that are able to be produced in the presence of the Lac repressor protein are unable to activate the T7 promotor, because the lysozyme produced by the pLysS degrades them before T7 promotor can be activated. However, induction with IPTG inhibits the production of the Lac repressor protein, so the cells have an abundance of T7 RNA polymerase, which are then able to activate the T7 promotor, and thus lead to the expression of rGFP. The aforementioned rGFP gene sequence contains the protein sequences for GFP, ampicillin resistance, and His6 and Xpress epitope tags. The GFP gene is a 238 amino acid long protein, with the amino acid residues, serine, tyrosine, and glycine, at positions 65, 66, and 67, respectively, that constitute its chromophore. The His6 tag is composed of 6 repeated histidine resiues near the N-terminus of the protein. The ampicillin resistance was incorporated into the plasmids to ensure that only bacteria that had incorporated that plasmid into their genome would be able to grow. Figure 2 has been provided to give a schematic representation of the rGFP protein sequence.
Therefore, after treatment with the <pLysS> and <pRSETA-GFP>, the G strain was then inoculated with IPTG, and 1 mL samples were collected at different times after inoculation. The G0 strain was collected immediately after IPTG was, so the post-inoculation time was 0 hours. The G3 sample was collected at 3 hours after IPTG induction. The rGCE, or rGFP crude extract, was isolated from the G3 sample and the rGCE was used to purify rGFP using Ni2+ agarose affinity chromatography.
Following the purification procedure, Bradford assay and spectrofluorometric data led to the total protein amount and specific activity estimates of the experiment. Figure 3 depicts the relative fluorescence and total protein amounts of the wash and elution fractions collected from the Ni2+ agarose column. The wash fractions had greater amounts of total protein than the elution fraction, with W3 having approximately 128 µg of total protein. However, the elution fractions had greater relative fluorescence values. E2 and E3 had the highest fluorescence at 88,000 and 84,000 RFUs, respectively. The specific activities, which is ratio of protein activity to total amount of protein, of E3, was calculated to be .
Additionally, the information obtained from the SDS-PAGE/Coomassie Blue analysis was used to estimate the protein purity and relative molecular weight of rGFP. Figure 4 is a labeled diagram of the SDS gel that was collected. Based on the molecular weight ladder that was loaded with our samples, the boxed band in the E3 lane was assumed to represent rGFP. A standard curve was generated from the distances traveled by the samples in the molecular weight ladder. Based on its distance traveled, the biphasic extrapolation line of the standard curve estimated the relative molecular weight of rGFP to be 36 kD. The purity of the rGFP was presumed to be 35%, because the rGFP band accounts for 35% of the intensity of all the bands in the E3 lane. Since its total protein amount was previously determined by the Bradford assay to be 30 µg with a purity of 35%, the yield of rGFP was calculated to be 10.5 µg.
The western blot, depicted in figure 5, provided conclusory evidence to determine if rGFP was present in the collected chromatography fractions. The western blot procedure involved probing the SDS-PAGE samples with anti-Xpress epitope antibodies. The anti-Xpress antibodies were only able to bind to the bands expressing rGFP, which were all of the bands except for the G0 band. Therefore, the western blot confirmed the presence of rGFP.
Conclusion/Discussion:
The series of experiments conducted successfully expressed and purified rGFP in the BL21 (DE3) <pLysS> <pRSETA-GFPUV> E. coli strain. Qualitative and quantitative measures confirmed rGFP activity. As expected, the elution fractions had higher activity and specific activity than the wash fractions. The higher specific activity values provide evidence that the procedures successfully isolated rGFP from the protein mixture. The wash fractions had higher amount of total protein, which was consistent with the predictions. The rGFP band for the elution fractions had a greater thickness than the other samples analyzed using the SDS-PAGE technology, which was also consistent with their higher purity and further corroborated that the rGFP purification procedures conducted were effective. However, the experiment conducted does have potential points of improvement. If an additional lysis procedure was conducted, the total yield of rGFP could have been increased. Sonication, which uses sound waves to burst cell membranes, could be compounded with the freeze-thaw method conducted in the experimental procedure to release more rGFP protein from the cells.
Additional experiments are needed to be able to unlock the potential scientific power of rGFP. Since the green fluorescence is caused by a cyclization reaction of three specific amino acids in the GFP chromophore, experiments could be conducted to test the effects of mutations to those three amino acids. The amino acid mutations could alter the cyclization reaction and thus change the excitation or emission wavelength of that unique form of GFP causing the protein to fluoresce a different color. Another pertinent follow-up procedure could include determining the temperature optimum of GFP. By testing GFP expression and activity in E. coli across a range of temperatures, the experiments could be used to determine what range of temperatures could GFP be used in to perform other investigations.
GFP has immense practical scientific application. By adding the GFP protein sequence at the end of specific protein sequences, GFP can be used to track the aggregation of proteins associated with specific diseases. The aggregation of specific proteins is suspected to cause Parkinson’s, Huntington’s, and other diseases, so GFP could be used to study where these protein aggregates deposit themselves and their effects on body functions (Willingham). Furthermore, GFP also has implications in cancer research. By expressing GFP in both cell lines, the activities of metastatic and non-metastatic cancer cell lines in blood vessels were compared (Hoffman, 2015). The metastatic cells lines were found to have superior attraction to the walls of blood vessels (Hoffman, 2015).
Bibliography:
Hoffman, Robert M. “Application of GFP Imaging in Cancer.” Laboratory Investigation, vol. 95, no. 4, 2015, pp. 432–452.
Pan J, Pickett E, Rippel S. Biochemistry Laboratory Manual. Richardson: University of Texas at Dallas, 2018. Print.
Willingham, Emily. “Why Do Jellyfish Glow?.” Nautilus, 5 Jan. 2017, nautil.us/issue/44/luck/-why-do-jellyfish-glow.