JACQUELINE+E

=Exploring the Expression, Purification, and Characterization of Purple Protein: pGEM-gbr22 =

====Protein expression is a subcomponent of gene expression. The expression of a recombinant protein in bacteria requires the insertion of a DNA fragment into an expression vector and the transformation of the bacterial cells. After this, the cells are cultured, induced to express the protein, and harvested by centrifugation. The objective of protein expression is usually to produce a sample that supports a certain biochemical or biological activity, such as enzyme catalysis or protein-ligand interactions. Frequently, it is not necessary to express the full-length protein to address a particular biological question. However, the choice of the N- and C-terminal boundaries is extremely important because even small differences can dramatically influence both solubility and expression of the protein [1].====

====By overexpressing the protein of interest in bacteria, proteins that are in low abundance in their native organism can easily be purified for crystallography, enzyme inhibition assays, etc. This technique also allows to study human proteins without having to use human tissues [2]. Protein purification is a series of steps intended to isolate a single type of protein from a complex mixture. This is done by breaking the bacterial cells to release the soluble proteins; by removing the insoluble cell debris by centrifugation; and by separating the desired protein from all other proteins using column chromatography. Then, the size and purity of the protein sample is determined by using gel electrophoresis (SDS-PAGE), a process called protein characterization [1].====

====This experiment consisted of overexpressing a recombinant protein in E. Coli BL21 (DE3) bacterial cells transformed by pGEM-gbr22. Then, the protein was purified and characterized. The hypothesis for this experiment is that E. Coli BL21 (DE3) bacteria will express the purple protein gbr22, which will be isolated from all the other proteins through purification.====

====In order to start the experiment, safety precautions were taken such as wearing gloves, goggles, and a lab coat. First, 25ul of bacterial cells and 1.597ul of DNA plasmid pGEM-gbr22 were added to a test tube and were heat-shocked. Then, 200ul of SOC media were added to the tube, which was incubated for 30 min at 37C and 250 rpm. After this, the cells were placed into agar plates and were incubated overnight. The next morning, a single colony was collected from the plate, transferred to LB/amp media, and incubated for 8 hours. In the evening, 0.625ml of starter culture, 25ml of LB, and 50ml of ampicillin were transferred to a 125ml Erlenmeyer flask, which was then incubated for 16-24 hours. The next day, bacteria was poured into a 50ml conical tube and was centrifuged for 10 min. Supernatant was decanted and pellet was weighed. Subsequently, 2.5ml of 1XPBS and 50ul of lysozyme were added to the 50ml tube containing the pellet and tube was stored in -20C freezer. In the second lab, the pellet was thawed, 2ml of Cyanase were added to it, and it was incubated for 10 min. Lysate was distributed into 1.7ml tubes and centrifuged for 20 min at 14,000 rpm at 4C. Supernatant was decanted and filtered with a 0.22 PES syringe filter. Column chromatography apparatus was set. Solution was mixed with 0.5ml Ni-NTA resin/buffer mix. Wash and elution buffer were used to isolate protein of interest. Nanodrop spectroscopy was used to determine the concentration of the protein. Lastly, the protein was characterized by gel electrophoresis using the 6 samples obtained during the first 2 labs.====

__ Results: __
















**Beer's Law Calculation:** Concentration determined of protein using 280 nm wavelength A=Ebc (0.32) = (38,850) (1) (c) c = 8.546 x 10 ^-6 mol/L (8.546 X 10^-6 mol/L)(25794.2 g/mol)= 0.2204 g/L or **0.2204 mg/mL** **Yield Determination:** At 280 nm wavelength (0.2204 mg/mL) (5 mL) = **1.102 mg**





====Lysozyme was added to the bacterial cells at the end of the protein expression lab since it digests the bacterial cell wall, which ruptures upon freezing. Cyanase was added since it digests DNA/RNA from the solution. The purpose of adding lysozyme and Cyanase is to release gbr22 protein in order to purify it using the HIS tag, which allows faster and efficient purification. The HIS tag works by modifying the protein to have six histidine residues added to the C-terminus, which can be easily used to separate the protein from other cellular proteins. The histidine residues will bind to cations like nickel that can be immobilized on a column matrix such as Ni-NTA. Then, the protein can be released from the Ni-NTA by adding imidazole, which is a competitive inhibitor for nickel with the protein.====

====Figure 10 shows the dried gel obtained from gel electrophoresis. Lane 1 is not showing anything since it was skipped to prevent gel distortion. Lane 2 is showing the molecular weight standard used in this lab (Figure 9). Lane 3 had sample 1, which contained E. Coli cells after they were induced to express gbr22 protein; therefore, a lot of protein bands are shown. Lane 4 contained sample 2, which had the soluble fraction of the protein, which is the reason of why there are so many protein bands too. Sample 3, which is everything that did not bind to the nickel, is shown in lane 5. Sample 4, collected after the wash buffer step, is shown in lane 6 and it has less protein bands due to the imidazole. However, fewer bands are shown in lane 7 and 8. This is because the samples from these lanes are samples 5 and 6 which had the elution buffer. Elution 1 was purple since it contained the gbr22 protein. On the other hand, elution 2 had a clear color since it only contained a very small portion of the protein. The difference between the elution buffer and the wash buffer is the concentration in imidazole since elution buffer had a higher concentration. In addition, the wash buffer removed proteins that were only loosely bound to the resin while the elution buffer released the gbr22 protein.====

====It was expected to observe gbr22 protein by itself in lane 5. However, this lane showed about 3 other protein bands besides gbr22. As a result, the purity of the protein was 30%. The molecular weight of gbr22 was approximately 25,700 kDa. Some possible sources of error include the incorrect creation of imidazole or of 10XPBS, not adding enough plasmid DNA, and not heating shock the bacterial cells long enough. In addition, the contamination shown in lane 7 and 8 might have occurred because there were other proteins containing rich regions of histidine that can bind to the nickel. ====

====E. Coli BL21 (DE3) bacterial cells were transformed using pGEM-gbr22, a plasmid DNA. A starter culture of bacteria was used to create a larger culture in order to express the protein gbr22. Then, the cells were harvested and frozen for the purification process. In the purification lab, the overexpressed protein was purified. This was accomplished by breaking the bacterial cells to release soluble proteins, by removing insoluble cell debris through centrifugation, and by using the affinity tag and Ni-NTA resin to isolate the protein of interest. The samples obtained from the first two labs were analyzed in the third lab using gel electrophoresis. This process leads to the future implications of enzyme assay where the activity of the protein is measured.====

====[1] Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards,==== ====A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.;==== ====Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.;==== ====Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.;==== ====Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.;====

====[2] European Molecular Biology Laboratory. Protein Expression and Purification Core Facility. [|http://www.embl.de/pepcore/pepcore_services/protein_purification/purification/index.] (accessed April 17, 2013).====