Expression and Purification of recombinant Green Fluorescent Protein (rGFP) from E.

coli using Ni+2 Agarose Affinity Chromatography

ABSTRACT: The purpose of this experiment was to determine if a His6 tagged recombinant form of GFP (rGFP) from the E.coli strain could be expressed and purified. This experiment began with purifying rGFP using Ni+2- agarose affinity chromatography. The samples from rGFP elution were quantified to determine relative fluorescence activity of GFP. The elution sample (E3) of fluorescence activity had 12446 RFU/mg. The total protein amount of rGFP fractions was determined by using a Bradford assay and a standard curve, and total protein amount in elution sample (E3) was 57.3 ug. The purity and molecular weight was determined by comparing intensity and location of bands to molecular weight ladder on an SDS-PAGE gel, which had 70 percent purity and 31.3 kDa (molecular weight). The western blot test confirmed rGFP .

INTRODUCTION: The Green Fluorescent Protein (GFP) was first discovered and isolated from the Aequorea Victoria jellyfish by Osamu Shimomura. Due to the chemical structure of the GFP, the chromophore is enclosed in an eleven stranded beta barrel which gives the ability to generate visible internal fluorescent. The cyclization interaction between Ser65 and Gly67 occurred in chromophore, and this interaction is responsible for its fluorescent. The electrons within the protein were excited at a wavelength of 395nm and that raises the energy of the protein. As the protein loses energy, green fluorescence is seen at a wavelength of 510nm. In order to observe better green fluorescent activity, the modified GFP (GFPUV) was used for this experiment instead of wild GFP. The purpose of histidine 6 tags in rGFP is to help purify rGFP using Ni+2-agarose affinity chromatography. Since histidine has a high affinity to bind with Ni+2, the role of histidine tag is to isolate rGFP from contaminant proteins. When the samples passed through the Ni+2 agarose affinity column, histidine tags in rGFP bind to the column and any contaminant proteins will be washed through the column. To elute the rGFP protein in the column, the elution buffer containing imidazole is used and competed with Histidine residues for the Ni+2 binding sites because structurally it looks like histidine. The purpose of this experiment was to determine if a His6 tagged recombinant form of GFP (rGFP) from the E.coli strain could be expressed and then purified with Ni+2-agarose affinity chromatography.

MATERIALS AND MEATHODS: Expression of rGFP Grow G bacterial culture, containing BL21(DE3)<pLysS><pRSETA-GFPUV > with the GFP sequence. Incubate 10ml of liquid LB growth media, containing 100ug/ml Amp, 25ug/ml Cam, with bacterial culture G at about 37oC until OD600 reach 0.5. Transfer 1ml of the culture into 1.5ml centrifuge tube and centrifuge to obtain pallet. The supernatant will be discarded and the tube, containing bacterial pellet, will be labeled G0 and stored at -20oC. Incubate the culture with IPTG (1mM) and allow to grow for 3 hours. At 3 hours post-induction, collect 1ml of the culture and centrifuge. The bacterial pellet will be labeled G3 and stored at -20oC. Additionally, collect 15ml of the G0 and centrifuge, and label it as G3-15ml and stored at -20oC. Preparation of rGFP Crude Extract (GCE) Add breaking buffer (10mM Tris, pH 8.0; 150mM NaCl) twice (total 1ml) to the G3-15ml frozen bacterial pallet. Immediately pipette up and down until the pellet dissolved. Transfer all the homogeneous solution to a centrifuge tube and vortex for 5 minutes and place in 37oC water bath for 10 minutes. Transfer the centrifuge tubes to a rotating platform shaker and incubate in a dry air at 37oC for 20minutes. After that, centrifuge the mixture at 14000xg, 4oC, for 10minutes. Transfer the supernatant into a new centrifuge tube and take a small sample, and the remaining supernatant will go through the Ni+2 agarose column. Preparation of a Ni+2-agarose column Place a small amount of glass wool into a 3 ml plastic syringe. Pipet about 100ul of breaking buffer into the syringe to remove air bubbles (Over flowing). While the buffer is flowing out, pour some breaking buffer into the luer-lock and then screw it onto the syringe. Add 2ml of additional buffer to the column, open the luer-lock, allow several drops of buffer to flow out of the column, and then close the luer-lock. Add 1ml of 50% Ni+2 agarose into the column and open the luer-lock to gravity pack the agarose matrix in the column. Add breaking buffer to the column to wash out the ethanol of the Ni+2 agarose to equilibrate the column and open the luerlock. Then close the luer-lock, and slowly apply the crude extract to the Ni+2 agarose column. Open the luer-lock and collect the 0.5 ml of the effluent in a centrifuge tube labeled W1 for wash #1. Add 0.5 aliquots of breaking buffer to the column and collect the washes in tubes W2-W10. After that, add 10 increments of 0.5ml of elution buffer, containing 10mM Tris, pH 8; 150mM NaCl, 300mM imidazole, and collect in tubes E1-E10. Determining total protein amount using the Bradford assay Mix the solution containing protein with water, and add Bradford reagent dye so that the total volume of the assay is 1.05ml. Vortex and incubate it for 10 minutes, and then transfer assay to a microtiter dish. Immediately read the absorbance at 595nm using a spectrophotometer. To

determine total protein amount of rGFP fraction in the samples, the Bradford assay must be compared to a Bradford standard curve. Bradford standard curve is created using six different known amouts of BSA: 0ug, 3ug, 5ug, 10ug, 15ug, and 20ug of BSA, and the standard curve data plotted on the graph is used to draw best-fit line of the data. Perform a Bradford assay, in singlicate, for the 12 samples W1-W6 and E1-E6. If there are samples whose absorbance reading fall outside the standard curve, repeat that assay on that particular sample. Once you determine what volume of sample to use for each fraction, repeat the Bradford assays two more times for each sample. At the end, the amount of total protein in the volume of sample is determined by extrapolating the absorbance value on the standard curve. SDS-PAGE/Coomassie Blue Analysis of rGFP Fractions Make a 12% resolving gel, containing water, 4x resolving buffer (0.75M tris pH 8.8, 0.4% SDS), 30% Acrylamide, 10% APS, and TEMED. Pour the 12% resolving gel into a glass plates and overload water to remove the bubbles. Pour the 5% stacking gel, containing water, 4x stacking buffer (0.25M tris pH 6.8, 0.4% SDS), 30% Acrylamide, 10% APS, TEMED), on top of 12% resolving gel and immediately insert a comb to form the walls. Prepare the samples with water and 4x sample loading buffer in centrifuge tubes and then vortex, boil, and centrifuge the samples: G0, G3, GCE, W2, W3, E2, E3, ladder samples. Put gel glasses and negative electrode chamber inside a tank, and then pour the electrophoresis buffer inside the tank. Load all the samples into lanes and electrophorese the gel at 200v until bromophenol dye front reaches bottom of green gasket. After that, strain the gel with Coomassie Blue dye. Western Blot Transfer of r GFP Fractions Make an SDS-PAGE gel using the same protocol that we have done previously. Transfer the protein from the SDS-PAGE gel onto nitrocellulose transfer membrane using gel holder eletrode cassettes, filter paper, and nitrocellulose membrane. After transferring protein, strain the membrane with Ponceau S strain for 2 minutes, and then rinse the nitrocellulose several times with ddH2O until bands appear on the membrane. To block the contaminant, place the membrane in a container containing 5% non-fat dry milk/TBS solution, and incubate the membrane on a shaking platform for 30 minutes. Discard the blocking solution and add 0.05% Tween 20/TBS and incubate the membrane on a shaking platform for 5 minutes. Repeat this wash process two more times. Add mouse IgG anti-Xpress epitope MAb solution and incubate the membrane on a shaking platform for primary probing step. Repeat the wash step as we have done previously three more times. Add Sheep IgG anti-mouse IgG conjugated horse radish peroxidase polyclonal anti-serum solution and incubate the membrane on a shaking platform for secondary probing step. Repeat the wash step two more times. Add only TBS and incubate the membrane on a shaking platform for final washing step. After that, add TMB substrate solution to the membrane and incubate it until visible band appear. When the desired color intensity is observed, stop the development process by transferring membrane into container with full of tap water.

RESULT: Figure 1 Expression plasmid map, a schematic diagram of the rGFP protin In the stain of E.coli, BL21(DE3) <pLysS> <pRSETA GFPUV>, lac repressor represses the lac promoter, which is needed for T7 RNA polymerase. T7 RNA polymerase, produced, in turn, binds to T7 promoter on pRSETA GFPUV plasmid to start gene expression and express the fluorescent of rGFP. Since there were not enough of expression in this way, so culture is induced with IPTG to release lac repressor from the lac promoter and increase rGFP production. G0 and G3 were bacterial culture of E.coli. G0 is the bacterial culture that was not induced yet, but G3 were induced with IPTG for 3 hours post-induction. W1-W10 were the wash fractions that fail to bind the Ni+2 agarose column using breaking buffer. E1-E10 were the elution fractions that bind the Ni+2 agarose column using elution buffer.

Figure 2 Combined elution profile From referencing Figure 2, E2 had highest fluorescent activity, 74797.5 RFU, indicating that E2 fraction has most of rGFP.

Figure 3 SDS-PAGE/Comassie gel SDS-PAGE gel was used to determine purity and relative molecular weight of rGFP. The gel was made with a 12% resolving gel and 5% stacking gel, and a comb was inserted to the gel to form the walls. 8 different samples that collected from the purification were loaded: G0, G3, GCE, W2, W3, E2, E3 and ladder. Ladder had 6 different standard molecular weights: 97.4KD, 66.2KD, 45KD, 31KD, 21.5KD, and 14.5KD. The band circled on stained gel indicated the rGFP and the molecular weight of rGFP was approximately 33KD. Wildtype GFP has a molecular weight for 27kDa. The percent of purity was determined by comparing the band intensity. From referencing Figure 3, the percent of E3 purity was approximately 70 percent. The total yield of rGFP was determined 40.11 ug for E3 fraction by multiplying 70 percent of purity to 57.3 ug of total protein amount.

Figure 4 Western blot Western blot was used to identify rGFP that react with specific antibodies. From referencing Figure 4, it had all bands of lanes GCE, G0, G3, E3 in the range of the molecular weight of 33KD, indicating rGFP existence. This result corresponds to the molecular weight of rGFP from SDS-PAGE/Comssie gel.

CONCLUSION/DISCUSSION SECTION: GFP that was expressed and purified is really important to science since fluorescent protein can be used as tracer inside cells. One promising application of fluorescent protein in biomedical research is to understand the cellular mechanism of brain disease. By using fluorescent protein, scientist can track the brain cell and give better understand how the brain cell interacts with other in people with brain disease, such as Alzheimers and Parkinsons diseases. However, since brain cell cannot be easily distinguished, brain cell should be illuminated by using different color of the fluorescent protein indicating use florescent proteins to give each cell a different color(Lichtman). GFP is also used in study of cancer. By using fluorescent protein, scientist can observe how cancer cell migrate in human body and exchange DNA. Once the tumor is placed on tissue inside human body, tagged rGFP cell help figure out where the placement of cancer cells are and help eliminate the issue before it escalates.