Green Florescent Protein: Properties and Purification for Scientific Experiments

Green Florescent Protein, abbreviated as GFP, is a protein composed of 238 amino acids that is commonly found in mnemiopsis, comb Jelly. It has a major wavelength at 396 nm and a minor one at 475 nm. GFP is what gives mnemiopsis their bright green florescent glow. ultraviolet light, or blue light, is necessary to see the florescent glow of this protein. GFP is an irregular protein because It Is highly resistant to denaturation by temperature and PH. It can survive In temperatures up to 98 degrees and has a pH of 12. 2 due to Its complex exterior, called the beta barrel. At an pH higher than 12. It denatures. It also has an Isoelectric point at 5. 3. The peripheral beta barrel cannot be digested or broken apart by protease because of the strong bonds holding It together. The beta barrel protects the chromophore, which Is the substance which gives GFP Its green glow. When CFP Is extracted from the plasmid of an E. Coll or from a Jellyfish, It contains an array different contaminants making it difficult for scientist to do experiments with GFP. A procedure in purifying GFP from a crude cell extract to nearly 100% GFP so that it can be analyzed and used in scientific experiments and research is necessary.

The goal is to ptimize each protocol used to purify crude GFP. Methods Ammonium Sulfate Precipitation To purify the crude samples of GFP, the ion exchange method separates substances inside the test tube by similar charge. A sample of crude GFP of 7. 5 mL in a plastic tube was used for the experiment. Knowing that 43. 6 grams of ammonium sulfate in a 100 mL solution yields a 70% percent saturated solution, the proportion 43. 6g 11 00 mL=x/7. 5 mL was used to determine that 3. 27 grams of ammonium sulfate needs to be added to the experimental sample. After adding the ammonium sulfate, the solution was stirred gently to prevent frothing.

Once most of the solution is transferred, the tube was placed on a triple beam balance along with another tube that went through the same process. The centrifuge was set at 15,000 rpms for 15 minutes so that the hydrophobic materials will separate and become the supernatant while the GFP pellet will remain behind. Once the 15 minutes elapsed, a new pipette was used to remove the supernatant, leaving behind the pellet of GFP and hydrophilic contaminates. To remove the hydrophilic substances, 5 mL of 4 molar ammonium sulfate and 15 mL of 10 mL tris at a p of 8 was added Into the oak ridge entrifuge test tube.

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The solution Is then put Into the centrifuge at 15,000 rpm for 15 minutes again. Once 15 minutes has passed, the supernatant, containing the GFP, was removed by a pipette and put In a microfuge. Hydrophobic Interaction Hgure yaropnoDlc Interactlon set up One molar ammonium sulfate was added to the column to wash the sample. Adding 1 molar ammonium sulfate washes the sample because a high salt concentration increases hydrophobicity of the GFP and the buffer, causing most of the GFP to be at the very top of the column. Substances that are hydrophilic get flushed out of the olumn while the more hydrophobic substances stay in the container.

After the column has been eluted with 1 molar ammonium sulfate, the tris buffer is added to the ammonium sulfate to dilute it into . 5 molar ammonium sulfate. The volume of 1 molar ammonium sulfate inside the oak ridge centrifuge test tube is the volume of the tris buffer that will be added. After the column chromatography has been flushed with . 5 molar ammonium sulfate, more hydrophobic substances will be flushed out since the hydrophobicity of the tris buffer and the GFP has decreased. This causes the GFP to spread out in the column. Finally the amount of . 5 molar ammonium sulfate is diluted with tris buffer to . 5 molar ammonium which should cause most of the GFP to be flushed out of the column along with other substances that are very hydrophobic. While this experiment is going on the liquid that comes out of the column is collected in multiple test tubes. These test tubes contain GFP and other contaminants. The solutions are than read by a spectrophotometer. Each test tube will be tested by the spectrophotometer so that a graph can be made. Anion Exchange Figure 2: Siphon Bridge set up for Anion Exchange Figure 3: Centricon Test Tube In order to use anion exchange, the starting condition of the sample needs to be in a low salt solution.

However after the GFP had gone through hydrophobic interaction, it was in a high salt solution. Before facing this dilemma, the fractions were pooled by centricon which decreases the overall sample volume by removing some buffer and salt solution. This greatly increases the GFP concentration in the samples. The fractions are placed in the centricon and then into a centrifuge for 25 minutes at 3,000 rpm to be separated by size. The large proteins stay in the entricon while buffer and salt solution goes into the plunger. To reduce the concentration of salt in the GFP sample, the sample is diluted 10 folds.

Since the amount of GFP that was restored was 18 mL, 162 mL of tris buffer needed to be added. The diluted GFP is then put in the chromatography container, containing positively charged DEAE which is attracted to the GFP at a low salt concentration. After the GFP has been completely filled, the column is connected to a beaker that contains a low salt concentration. the low salt concentration beaker is connected to a high salt concentration beaker. As one drop of low salt solution goes into the chromatography column, one drop of high salt solution goes into the low salt solution.

Gradually the salt concentration increases in the low salt beaker and in the column chromatography, causing GFP to spread down the container. The eluted GFP dripped out of the column chromatography to be collected in test tubes. I nree pnase partltlonlng Figure 4: Precipitate of GFP. T-butanol is one top while contaminates are on bottom GFP then went through three-phase partitioning, also known as TTP. The fractions taken after an anion exchange was 15 millilieter. Ten ml of 4 M ammonium sulfate was added to this volume to increase the salt concentration of the solution to 1. M, which is about 40% salt saturation. Twenty-five milliliters of t-butanol was added then added which was the same amount of ammonium sulfate and GFP in the container. The container was then placed in the centrifuge for ten minutes at 4600 RPM, causing the mixture to split into three layers; butanol on top, GFP in solution on the bottom, and precipitated contaminants in-between. The top layer of butanol and disk of precipitate were taken out. The volume of GFP solution was again matched in utanol and the container went into the centrifuge again. An aspirator was used to extract the GFP into a microfuge. . 6M ammonium sulfate was then added to the microfuge and the container was placed in a micro centrifuge for one minute at 13,000 RPM. Butanol and other contaminants that had not been take out previously formed a disc, was then taken out with an aspirator and the remaining GFP was then left in the microfuge. HPLC Figure 5: HPLC basic layout After the sample went through three phase partitioning, it was put through the High Performance Liquid Chromatography for a final purification. First liquid was put into the HPLC to clean out any previous GFP inside the loop of the HPLC and the column of the HPLC.

Then, GFP in the microfuge was sucked into an injector to be put into the HPLC. Pushing the top of the injector slowly, GFP entered into a loop inside the HPLC. Once the GFP was placed in the loop, a knob was turned clockwise to the word lock. The GFP was then sent to the column where it was purified further by size through the minuscule beads. About 6,000 pounds of pressure per square inch was produced by the HPLC to push the GFP through the beads. While this was occurring, a pectrophotometer connected to the HPLC read the wavelengths of substances.

Near the 396 nm wavelength, GFP was collected in a microfuge tube. A UV light was held near tne exlt 0T Results e HPLC to measure tne amount ng sample. Graph 1: Results of the sample after HIC at a wavelength of 395 nm Graph 2: Results of the sample after HIC at a wavelength of 280 nm Graph 3: Results of the sample after HIC of the entire spectrum Seventeen test tubes were received after the HIC purification process. A blank consisting of tris buffer and ammonium sulfate was sampled in the spectrophotometer against liquid from each of the seventeen test tubes.

Graph one represents the sample after HIC at a wavelength of 395 nm while graph two Results shows the results after HIC at a wavelength of 280 nm. After HIC, the fractions 12 to 16 were chosen for their purity and recovery of GFP. Graph one show the amount of GFP in each fraction number while graph two shows the total amount of protein in each fraction number. Graph three shows the spectrum of the entire sample. Graph 4: Results after Anion Exchange at a 397 nm wavelength Graph 5: Results after Anion Exchange at a 280 nm wavelength Graphing 6 Thirteen test tubes were collected from the Anion Exchange purification process.

This time the samples were blanked against tris buffer at 8. 0 pH and 0. 5 molar sodium chloride. Graph four shows results of the Anion Exchange at a 397 nm wavelength and graph five shows the results after Anion Exchange at a 280 nm wavelength. Once again, the graph at a 297 nm wavelength shows the amount of GFP while the graph at a 280 nm wavelength shows the amount of total protein. Graph six represents the results of the entire spectrum. The GFP peak was a lot more visible. Step Iotal sample (mL Abs (280) Total Protein Abs (397) GFP Ratio Crude sample 120 1600 . 25 At-ns042- 20 1 . 61 . 9 118 HIC 18 . 28 . 173 . 618 15 . 126 . 130 1. 03 3 Phase Partitioning . 01 n/a . 75 . 243 . 257 1. 06 Table 1: This is the overall data table. The second column shows the total volume at the start of each purification step. The following two columns are the peaks of the graphs at those wavelengths. The last column represents the ratio of GFP to the total Protein. The most desirable ratio is 1. 25. Dlscusslon The first method in purifying the crude GFP was using the ammonium sulfate precipitate. When ammonium sulfate is placed in water, it dissociates into ammonium (NH4+) and sulfate ions (S042-).

Water, composed of two hydrogen ions and one oxygen ion, is a polar molecule because the oxygen has a high electronegativity. Oxygen has a greater affinity making the oxygen portion of water negative and the hydrogen portion of the water positive. The dissociated positively charged ammonium ion is allured to the negatively charged oxygen while the negatively charged sulfate ions are attracted to the dissociated positively charged hydrogen. The attraction between the ammonium sulfate and the water was so strong that the GFP and other proteins were left unoccupied, causing them to precipitate.

When GFP in the 70% salt solution was placed into the centrifuge, substances such as DNA and RNA was removed because they became part of the supernatant. At a 70% salt concentration, only hydrophilic substances stay in solution while the more hydrophobic substances precipitate. When the GFP in a 25% solution of salt was placed in the centrifuge, the GFP and other substances went back into solution because there not enough water was occupied by the salt. Before the GFP is placed in the centrifuge, it must be balanced with another centrifuge with the same weight and the two containers must be placed across from one another.

This is vital because the centrifuge needs to be balanced when it is rotating at an incredibly fast speed. Failure to have balanced centrifuge containers can result in a broken centrifuge and loud sounds. Also when mixing the GFP with salt, it is important not the shake the container or frothing will occur, making it difficult to transfer the solution in to an oak ridge centrifuge tube. The second purification procedure that GFP underwent was hydrophobic interactions. During this purification, GFP binded to the non-polar Phenyl Sepharose beads because of its non-polar and hydrophobic traits.

However the water in tris buffer is strong enough to separate the attraction between GFP and the Phenyl Sepharose. Therefore a high salt concentration is necessary to occupy the water so that the GFP and the Phenyl Sepharose to be attracted together. At a high salt concentration, GFP with bind easily to the Phenyl Sepharose since very little water molecules would interfere with the attraction and at a low salt concentration, GFP would not bind easily to the Phenyl Sepharose because tnere wlll De a lot 0T unoccuplea water molecules tnat wlll De aDle to InterTere wltn the GFP and Phenyl Sepharose attraction.

Before the experiment, ten millimolar tris buffer at a pH of 8 was used to clean the column in order to keep the pH stable and to wash away the salt, ammonium sulfate, in the column. Removing the salt is vital because the buffer that once surrounds the salt will be allured to the hydrophobic benzene and to the hydrophobic patches on the GFP. Since the hydrophobic patches of the GFP are already filled, they will be flushed out, leaving mostly beads of benzene and the 10 millimolar tris buffer at a pH of 8. Once the column has been clean, it needs to be equilibrated so that the salt concentration is the same through the olumn.

The step gradient used, started ata 1 molar ammonium sulfate concentration and was halved until a . 25 molar concentration to separate substances by hydrophobicity. The third purification procedure was anion exchange. In this procedure, GFP and other contaminants are separated by charge. The beads in the containers are different from the beads from the hydrophobic interaction because on they have a different chemical called DEAE which makes them positively charged. GFP has both protons and electrons on it which is why it was not easily attracted to the DEAE, which is why the GFP is put in a basic solution.

Ata high pH, the amount of negatively charged hydroxide increases and these hydroxides are allured by the protons on the GFP. The protons are than neutralized, making GFP a negatively charged molecule. The isoelectric point of GFP is at a pH of 5. 3. Ata pH higher than 5. 3, it is negatively charged and when it is at a pH lower than 5. 3, it is positively charged. Once the column chromatography is filled with GFP and connected to a beaker of low salt which connected to a beaker of high salt, anion exchange occurs. As the salt concentration increases, the GFP slowly spreads down the column and eventually out f the column into test tubes.

Between the HIC and the Ion exchange chromatography, the sample the fractions were pooled and put in a centricon causing the GFP concentration in the samples to increase. This occurred because the ultrafilter only allowed particles smaller than protein to go in to the pusher. The large proteins stay in the centricon while buffer and salt solution goes into the plunger. The sample of GFP was also diluted 10 folds because the sample needs to be in a low salt solution to use anion exchange and after the GFP had gone through hydrophobic interaction, it was in a high salt solution.

The anion exchange method creates a continuous salt gradient because as one drop of low salt solution goes into the column chromatography, causing GFP to spread down the container. The follow procedure was the three phase partitioning purification. T-butanol and 1. 6 molar ammonium sulfate were essential for this procedure. T-butanol has a low density causing in to stay above the GFP solution. In addition it has an attraction for water and other hydrophobic substances causing 5 mL of water to be drawn out of the GFP sample and precipitated substances to float between the t-butanol and the GFP sample.

Fresh t-butanol is necessary after removing the old t-butanol with the contaminants because at that point, the salt concentration had increased since water was drawn out. was aDle to De preclpltatea Decause 0T tne nlgn salt concentration. The final procedure for purifying GFP was using the HPLC which separated substances by size. The beads used in the HPLC column are miniscule and porous. The pours on the beads give substances of the same size more opportunities to leave the HPLC at the same time. Since the beads are so small, high pressure is needed to push the GFP sample through the beads.

Naturally, smaller substances will exit the HPLC first while larger materials will exist last. In all scientific experiments room for error is unavoidable. During the HIC, IEX, three phase partitioning, and the HPLC, amounts of GFP were lost due to the GFP sticking to a container, a pipette, and even spills. During the HIC some of the GFP was lost due the overflowing the test tubes with liquid exiting the column. During the HPLC some GFP was lost because not all GFP dripping out of the HPLC went in to the microphage. Other errors include letting the column dry because the liquid was not dded to the beaker about the column.

During the spectrophotometer runs, the blank was no inserted correctly causing the reading of the GFP to be incorrect. In addition, the order in which the GFP samples were suppose to be placed in the spectrophotometer was messed up. Judging from the overall purification table, table 1, the purification was quite successful. Originally, the ratio was only . 25, but by the end of all the purification procedures, it obtained a ratio 1. 06. A 1. 25 ratio is most desirable and through the purification, the ratio was nearly reached. The anion exchange, three phase artitioning, and the HPLC purification were the most impacting procedures.

The anion exchange greatly increased the purity of the crude sample compared to the HIC purification. The three phase partitioning and HPLC purified the GFP even more. Some improvements to the protocols would be to start with the anion exchange purification so that overall, the salt solution would go from a low salt concentration to a higher salt concentration. This also eliminates the need to dilute the solution. In addition, an automatic machine could be used to shift the test tubes that collect the iquid exiting the columns to prevent overflowing test tubes and the risk losing GFP.

GFP is unique because of its florescent glow. This glow can be used as a marker or an indicator. If a glowing marker could be placed on infectious cells such as tumor cells or cancerous cells, it would revolutionize the treatment of these diseases because doctors will be able to track where the harmful cells are. In addition, if it is possible to trigger the florescence of GFP with UV light, it can eventually be used in light bulbs to produce light. GFP light bulbs would last for an incredibly long time ince they are very resistant to denaturing.

In addition, in vehicles, GFP can be mixed in the motor oil, transmission oil, power steering oil, air conditioning oil, and other oils so that if a leak occurs in a car, it can easily be spotted by shinning UV light on the car. The purification of GFP can lead to endless new innovations in electrical engineering, automotive repair, and curing deadly diseases.

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