Shane+A

__ Finding the Purple Protein __

__ Introduction: __

Due to the increase and variety in modern technology, the usage and production of recombinant proteins in scientific research has increased greatly. One method of expression, especially for intracellular expression, includes "nickel affinity, ion exchange, and gel filtration purification" [1]. This method is very similar to the method used. The purity of the protein also depends on how strong the affinity for the ion is. "The final purity of the protein can be optimized by controlling the ration of recombinant protein to the colummn size; lower-affinity contaminants can be competed with a relative excess of the histidine-tagged recombinant protein" [2]. The goal of this lab was to isolate the purple gbr22 protein from the transformed E.//coli// BL21 host bacteria. The protein was purified and removed, as long as the bacteria is centrifuged and its components are separated properly.

__ Materials & Methods: __

In the first of the three parts of this lab, 25ul of bacteria (BL21(DE3)) were each added to the transformation tubes. Then 2ul of plasmid, which was pGEM-gbr22, was added to the bacteria. The tubes were heat shocked in order for the bacteria to take up the plasmid, followed by 2 minutes of bacterial recovery on ice. SOC media was added for bacterial growth and the bacteria was then grown on the Agar Amp plates. The next day, ampicillin was added to LB. A few colonies of the bacteria grown overnight were added to the LB. A few hours later, LB and ampicillin were added to a flask along with BL21(DE2) and then left on the shaking incubator. The next day, bacteria was poured into a conical tube and centrifuged for 10 minutes at 5000 rpm at 4 degrees Celsius on the Allegra X-15 benchtop centrifuge (Beckman Coulter, Inc., Brea, CA). 1xPBS was added to the cell pellet and vortexed in order to resuspend the cells in Phosphate buffered saline followed by lysozyme. In part two, 2ul of Benzonase (Sigma-Aldrich, St. Louis, MO) were added in the 50mL conical prepared in part 1. Lysate was clarified by centrifuging for 20 minutes at 14,000 rpm. The wash and elution buffers were then prepared. The lysate was syringe filtered through a PES syringe filter (Membrane Solutions, Plano, TX). Half a milliliter of Ni-NTA resin/buffer was added and mixed to the supernatant. An Econo column (Bio-Rad, Hercules, CA) was used to run the supernatant with Ni-NTA resin/buffer through. Waste, wash, and Elution 1 and 2 were collected. The Ni-NTA was stripped with 10 cv water, 10 cv .5 M NaOH, and 10 cv water again. During the Nanodrop spectrophotometer portion, absorbance of Elution 1 was recorded through a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, ED) at 280 and 574 nm and then converted into mg/ml. During the final portion of the lab, the protein was characterized. A 6x gel-loading buffer was used to prepare the samples. The tubes were placed on a heat block for five minutes and centrifuged at 5000 rpm for five minutes. An SDS electrophoresis was run for 25 minutes at 200 volts. The first well had the protein ladder (Thermo Fisher Scientific, Waltham, MA), along with samples 1-6 and 4-6 of the partner's. The gel was stained, covered with cellophane, and then dried.

__ Results: __

Figure 1: The E.//coli// BL21 colonies expressed a gene which was resistant to ampicillin (pGEM-gbr22). The protein expressed gives the bacteria a purplish color. Since the plate was treated with ampicillin, bacteria that did not express the protein were killed. The bacterial plate was incubated at 37 degrees Celcius for 24 hours.



Figure 2: The E.//coli// BL21 colonies aren't expressed on the "control" plate. Since they don't have the pGEM-gbr22 plasmid, they aren't ampicillin resistant, thus causing no bacteria to have grown on the plate. Figure 3: E.//coli// BL21 bacteria that expresses pGEM-gbr22 in a solution of 25ml of LB broth and ampicillin after being shaken at 37 degrees Celsius for 24 hours.

Figure 4: E.//coli// that expressed the protein were centrifuged using the Allegra X-15 benchtop centrifuge into a pellet form. The supernatant was leaked. The pellet weighed .16 grams.

Figure 5: Elution 1 was used to collect most of the purified protein.

Figure 6: Elution 2 was used to collect the rest of the purified protein from the column that Elution 1 wasn't able to collect.

Figure 7: Nanodrop spectrophotometer absorbance spectra for Elution 1 sample (with pGEM-gbr22) at 280 nanometers. Absorbance is equal to the molar absorptivity/extinction coefficient times the path length times the concentration (A=Ebc). The extinction coefficient for the purple protein is 118300 1/M*cm, the absorbance being .538, and the molar weight being 25794.2 grams. Thus, the concentration of the protein at 280nm is .013mg/ml.

Figure 8: Nanodrop spectrophotometer absorbance spectra for Elution 1 sample (with pGEM-gbr22) at 280 nanometers. Absorbance is equal to the molar absorptivity/extinction coefficient times the path length times the concentration (A=Ebc). The extinction coefficient for the purple protein is 118300 1/M*cm, the absorbance being .573, and the molar weight being 25794.2 grams. Thus, the concentration of the protein at 280nm is .0125mg/ml.

Figure 9: Nanodrop spectrophotometer absorbance spectra for Elution 1 sample (with pGEM-gbr22) at 574 nanometers (maximum absorption wavelength). Absorbance is equal to the molar absorptivity/extinction coefficient times the path length times the concentration (A=Ebc). The extinction coefficient for the purple protein is 118300 1/M*cm, the absorbance being .82, and the molar weight being 25794.2 grams. Thus, the concentration of the protein at 574nm is .179mg/ml.

Figure 10: Nanodrop spectrophotometer absorbance spectra for Elution 1 sample (with pGEM-gbr22) at 574 nanometers. Absorbance is equal to the molar absorptivity/extinction coefficient times the path length times the concentration (A=Ebc). The extinction coefficient for the purple protein is 118300 1/M*cm, the absorbance being 1, and the molar weight being 25794.2 grams. Thus, the concentration of the protein at 574nm is .218mg/ml.

Figure 11: SDS gel electrophoresis done at 200V for 25 minutes. The gel was then stained for an hour. After staining, the gel was dried. The top row is the ladder. The six rows below it are Samples 1-6 from one partner. The next three rows are Samples 4-6 of the other partner's. The purple bands represent the different proteins found. Sample 5, which was elution 1, had the largest concentration of the purple protein.

Figure 12: Molecular weights ladder used as reference in gel electrophoresis.

__ Discussion: __

The protein purified was the protein sought after. According to gel elctrophoresis, the purified protein from Elution 1 and 2 was the gbr-22 protein due to the stains being on the same row as each other, thus indicating the same protein. Lysozyme was added in Part two of the lab in order to digest the cell walls, allowing the proteins and other molecules of the bacterial cells to be exposed. Benzonase is a nuclease that reduces the viscosity by digesting the DNA/RNA in the mixture, allowing for the supernatant in the centrifuged solution to contain the proteins and not the other cell debris. The high molecular weight of the DNA causes the solution to be viscous, which is why the Benzonase is so crucial. Sample one contains the bacteria itself. Sample 2 contains the supernatant after the lysate was added. Sample 3 contains the waste from the column. Sample 4 contains the wash from the column. Sample 5 contains Elution 1, and sample 6 contains Elution 2. The wash cleared out all the excess cell debris left behind by the waste from the column, whereas the Elution buffer caused the actual purified protein to wash out. The HIS tag system works by causing the protein being searched for to bind to the Ni-NTA. This caused all the excess proteins and cell debris to be washed away by the other solutions. When the Elutions are used, the Imidazole caused the protein to detach from the Nickel and thus wash out, allowing the container to have mostly the purified protein sought after. Based off of the gel electophoresis, the gbr22 protein is about 24kDa. Sources of error include not including the proper concentrations of Imidazole in the Elutions, thus causing the protein to not strip off the Ni-NTA. Other sources of error include several cases of human error, which cause the protein to not be removed from the cell due to faulty centrifuging or disturbing the pellet.

__ Conclusions: __

This three part lab was performed in order to show the lengthy but efficient procedures of purifying a specific protein and finding different data from it. Recombinant DNA was inserted into E.//coli// to cause them to produce the designated protein. Data was collected from different instruments, and by being able to find the wavelength at which the largest amount of protein was produced, variables can be adjusted to the scientists advantage. The dried gel showed the approximate weight of the protein, giving the researcher confidence in procedure. Future directions and applications for this lab are to purify harmful proteins in other bacteria and to analyze them in order to find ligands to shut off either protein production or function.

__ References: __

[1] Gjerstorff, M.F.; Besir, H.; Larsen, M.R.; Ditzel, H.J., Expression, purification and characterization of the cancer-germline antigen GAGE12I: a candidate for cancer immunotherapy. //Protein Expr Purif//. **2010**, 73(2): 217-22.

[2] Graslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; Protein production and purification. //Nature Methods//. **2008**, 5(2): 135-46.

  