Please note! This essay has been submitted by a student.
The purpose of this experiment was to express and then purify a His6-tagged recombinant form of GFP from the E. coli strain given (BL21(DE3)) using the Ni+2 agarose affinity chromatography. Using the strain given, rGFP crude extract was first purified using Ni+2-agarose affinity chromatography. Next, the Bradford Assay was used in order to determine the specific activity which was found to be 179.5 RFU/mg. Then a SDS-PAGE stained with coomassie blue was used to estimate purity and relative molecular weight, which were ~50% and ~35kD respectively. Finally, a western blot was performed in order verify that rGFP was present.
Green fluorescent protein, also known as GFP, was first discovered in a species of jellyfish, the Aequorea victoria. Osamu Shimomura was the first one to discover this and was also the first person to isolate GFP and find out which part of the GFP causes it to fluoresce. The chromophore located inside the 11 stranded beta barrel is what caused the GFP to show a bioluminescent property, which was first characterized by Shimomura in 1979.1 The wild type GFP contains an open reading frame that codes for a 238 amino acid protein and has a relative molecular weight of ~27kDa. In rGFP, the recombinant version of GFP in E. coli, a His6 tag along with an X-press epitope tag is found on the N-terminus of the protein. The His6 tag is important because it allows for the rGFP to be purified through the Ni+2-agarose column because the rGFP should bind the column because of the interactions between the His6 tag and Ni+2.
The purpose of this experiment was to express and then purify a His6-tagged recombinant form of GFP from the E. coli strain given using the Ni+2 agarose affinity chromatography. Through this, the specific activity, purity, molecular weight, and the presence of rGFP can be found.
In order to prepare the GCE from an rGFP crude extract, rGFP was isolated from the given G3-15ml bacterial pellet. Breaking buffer was added twice to the conical vial with the G3-15ml bacterial pellet and was immediately pipetted up and down until the pellet was thawed. The homogeneous solution was then pipetted into a centrifuge tube and vortexed for 5 minutes and placed in 37°C water bath for 10 minutes. The centrifuge tubes were then placed on a rotating platform shaker in a dry air 37°C incubator for 20 minutes and then was centrifuged at 14,000xg, 4°C, for 10 minutes. The supernatant was then decanted into a new centrifuge tube labeled GCE. Preparing a Ni+2-agarose column The Ni+2-agarose column was set up by placing a small amount of glass wool into a 3 ml plastic syringe. The syringe was then clamped to a ring stand and the luer-lock was attached. With around 500ul of breaking buffer was added on top of the glass wool so that the bed volume was 0.5ml of bed volume, 1ml of 50% slurry of Ni+2-agarose was pipetted into the column and the column was allowed to become packed and pre-equilibrated.
With the GCE from the centrifuge tube, around 1ml of GCE was added to the Ni+2-agarose column. After 10 minutes, in 0.5ml increments, the GCE was collected in 10 centrifuge tubes labeled W1-W10. Elution buffer was then added to the Ni+2-agarose column and collected in 0.5ml increments of elution buffer in centrifuge tubes labeled E1-E10.
Six different amounts of BSA were added to a microtiter well and then brought up to a volume of 50ul with water. Bradford dye was then added to each sample and once the samples were in the microtiter well, they were incubated at room temperature for 10 minutes. Once they were finished incubating, the samples were then measured for their absorbance at 595nm and a standard curve was generated from the data (Absorbance vs BSA).
The 12 samples, W1-W6 and E1-E6, in singlicate, were put through a Bradford microplate assay three times. Once the absorbance values from the samples were found, the amount of total protein in each sample was determined by interpolating the absorbance value on the Bradford standard curve.
Mixing water, 4x resolving buffer, 30% Acrylamide, 10% APS, and TEMED, the 12% resolving gel was made and poured between two glass plates. The 5% stacking gel was then made mixing water, 4x stacking buffer, 30% Acrylamide, 10% APS, and TEMED and then poured on top of the 12% resolving gel with the comb being slid in the gel after.
Samples of G0, G3, GCE, W2, W3, E2, E3, and a ladder were then prepared with varying amounts of water and 4xSLB and loaded onto the gel in the lanes created by the comb. The electrophoresis tank was then set up and the samples were electrophored at a constant voltage of 2oo volts for around 45 minutes until the dye reached the bottom of the green gasket. Once the dye had reached the bottom, the tank was disassembled and the gel was observed to estimate the protein purity and relative MW.
A SDS-PAGE was prepared in the same way as before, using the same recipe for the 12% resolving gel and 5% stacking gel. Samples of GCE, W2, W3, E2, E3 were then prepared for Western blot and mixed with 4xSLB. The electrophoresis tank was then set up samples were loaded at a constant voltage of 200 volts for approximately 45 minutes. Once finished, the gel was then placed inside a transfer cassette with a nitrocellulose membrane.
The nitrocellulose membrane was removed from the western blot sandwich and was placed in a Tupperware container where Ponceau S stain was added. The container was then incubated with a rocking motion for approximately 2 minutes and once that was finished, the nitrocellulose was rinsed several times with ddH2O until red protein bands appeared. The MW ladder was then marked with a pencil. The nitrocellulose membrane was then placed in another Tupperware container with a lid and the blocking solution (5% non-fat dry milk/TBS/0.05% Tween 20) was added and the membranes were allowed to incubate on the shaking platform for 30 minutes. The blocking solution was then discarded and 7ml of mouse anti-Xpress epitope Mab solution was added. The membrane was allowed to incubate on the platform for a total of 45 minutes, being flipped every 15 minutes. The primary antibody solution was then poured out and TBS/0.05% Tween 20 was added to the container and left to incubate the membrane for 5 minutes. The wash solution was then discarded and this wash/incubation process was repeated two more times. 7ml of Sheep anti-mouse IgG conjugated horse radish peroxidase polyclonal anti-serum solution was then added and allowed to incubate the membrane for 45 minutes, being flipped every 15 minutes. Another washing step was then performed using the TBS/0.05% Tween 20 three times. Using the same container from the Ponceau staining, the container was filled up with water. TMB substrate solution was added to the membrane and allowed to rock until the desired color was achieved. Once the desired color was achieved, the membrane was then transferred to the container with water in order to stop the development process.
The expression of rGFP in E. coli starts with the IPTG inhibiting the Lac repressor which then allows the T7 RNA polymerase to bind to the T7 promoter. This binding of the T7 RNA polymerase and the T7 promoter activates the open reading frame and causes the transcription of the recombinant GFP. The ampicillin resistance is there to help maintain resistance from different types of contaminants and to help maintain selection for the plasmid. This process can be seen in the plasmid map. From the constructed schematic diagram figure of rGFP, it can be seen that rGFP contains a His6 tag, which is located on the 5’N terminus side, an Xpress epitope tag, which is located between amino acids 24-32, a chromophore, which is located between amino acids 103-106, and a 3’C terminus. The GFPUV DNA sequence can also be seen about 238 amino acids long on the 3’C terminus side of the rGFP. The combined elution profile of washed 1-6 and elutions 1-6. It can be seen that the wash with the largest RFU value was W4, with a RFU value of 6,792. The elution with the largest RFU value was E3, with a RFU value of 92,937. Using the sample which had the highest RFU value (E3), the total amount of protein calculated and obtained was 5.04ug. Using the RFU value and the total amount of protein, the specific activity found for the E3 fraction was 179.5.