KARUNA+A

__ Title: __ Gbr22 Protein Expression, Purification, Characterization in E.Coli Bacteria

Intro is not very clear, karuna.

__ Introduction: __ E. coli bacteria are used to be infused with plasmid vectors from various proteins due to the difficulty of expression in human tissues. The purpose of their use is the wide range of proteins that can be expressed in the cells. The growth is followed by application of lysozyme, which eliminates unnecessary debris, and the protein targeted is then filtered. This leads to purification specific to the protein at hand. Tags can be added (such as the 6xHIS tag used in this set of experiments) to separate unnecessary proteins [1]. The purity must be tested to ensure that the protein resulting from the process is not contaminated.

This series of tasks, expression, purification, and characterization, allows biological research to be facilitated. Proteins gained from this methodology can be used for testing different drugs or compounds. Active site compatibility can cause compounds to move further and advance to labs, perhaps becoming drugs for major epidemic diseases. The BL21 (DE3) E. coli bacteria express the pGEM-gbr22 protein in this set of labs. A plasmid selected from a purple coral residing in the Great Barrier Reef in Australia was used due to its resistance to ampicillin. The vivid hue facilitates locating the protein during the duration of the lab. Once the plasmid and bacteria are fused, the bacteria should be slowly separated away until the protein is isolated and purified as much as possible.

__ Materials & Methods: __

The first step in expression of DNA was to fuse the bacteria and plasmid. E. Coli bacteria and the plasmid DNA were inserted in tubes, incubated, and placed on agar dishes for overnight incubation. Three plates were used: DNA infused, control with no DNA, and a fun plate. A culture of purple bacteria grew in the DNA infused plate overnight, from which a sample was taken, harvested, and centrifuged. The pellet remaining at the bottom of the solution was preserved. After suspending the cells, lysozyme was added to break down cell materials. The goal for purification was to isolate the purple protein from other E. Coli junk material. Digested cell materials were incubated and cyanase was added to reduce viscosity and digest more materials. This solution was suspended and centrifuged to remove debris. Using a syringe filter, large particles were washed out of the protein solution. The Ni-NTA buffer/resin was added to the supernatant, after PBS was administered to clean the syringe tube. Wash buffer was inserted (low imidazole) and loosely bound proteins were washed out. Then elution buffer with high imidazole was used to release the protein. The Nanodrop spectrophotometer was used to find absorbance of elutions in two trials at 280nm. Then it was taken at maximum absorbance (574 nm). This process helped figure out the yield. Samples collected during purification were used to determine purity using the SDS-PAGE, which separated proteins by molecular weight. The samples were prepared for the gel using heat shock. Once the SDS-PAGE was loaded with the gel cassette, a loading buffer cleared out the wells. Then the treated samples 1-6 were inserted, followed by another 4-6. After the machine ran, the gel was washed thrice, stained, and dried. The resulting image helped determine protein purity.

__ Results: __

Figure 1A. Agar dish with Amp-positive, DNA (DE3), and plasmid pGEM-gbr22 after incubation for 24 hours at 37 degrees Celcius, cultures present as small purple spots. Figure 1B. Agar dish with Amp, BL21(DE3) DNA not present (control dish), incubated for 24 hours at 37 degrees Celcius, no cultures present.



Figure 1C. Dish with bacteria from keyboard and mouse rubbed on agar, small spots of colonies present, incubated at 37 degrees Celcius for 24 hours. Figure 2. Bacterial culture of BL21 (DE3) transformed with DNA plasmid of pGEM-gbr22 after incubation, from bacterial colony shown in 1A.



Figure 3. Pellet of protein cells formed from centrifuging BL21(DE3) culture

Figure 4. Elutions 1: 5ml gbr22 washed with 1xPBS and 250mM imidazole elution buffer (purple protein inside) Elution 2: 5ml gbr22 washed with 1xPBS and 250mM imidazole (clear due to lack of protein)

Figure 5. Elution 1 Run measured at wavelength 280, absorption .47 (by Nanopure Spectrometer) Figure 6. SDS-PAGE results of samples from throughout protein labs. Lanes flipped, first four being samples 6, 5, 4, and remaining six contain samples 6,5,4,3,2,1. Last lane is PageRuler protein ladder for reference.



Figure 7. PageRuler key for molecular weights of proteins.

__Calculations__ A=ebc Maximal Wavelength-574nm 0.044 = 118300 M-1cm-1 (0.1cm)(C C=.37194 mg/mL  Yield=.37194 mg/mlx5 = 0.18597

280nm 0.75 = 38850M-1cm-1 (1cm)(C) C = .19305mg/ml Yield= .19305mg/ml x5= .96525

The 280nm only accounts for certain amino acids such as tyrosine and tryptophan, which is why the outcome for the maximal wavelength, or 574nm, should be more accurate. It measures the entire protein instead of concentrating on certain amino acids.

__ Discussion: __ In the expression phase, the bacteria were successfully fused with the plasmid using temperature change techniques which caused the cell membranes to allow the DNA inside. After returning to optimal conditions, the bacteria continued to grow on the agar plates with the protein expressed. Sources of error included prolonged heating of the substances and accidental contamination. During purification, lysozyme and cyanase were used to digest cell debris such as the cell walls and membranes from the bacterial cells. The most important portion was the binding of Ni-NTA resin/buffer and 6xHIS tag. The nickel and protein combined were too large to be washed out until the high concentration of imidazole was used, which competed for the binding of nickel. Once the protein was detached it could be successfully collected in the Elution 1 tube. Elution 2 came out as it should have, clear, as there was no protein left in the syringe. The difference between wash and elution buffers is that the was buffer removes loosely bound proteins, while the elution has enough imidazole to release the NiNTA from the gbr22 protein. Sources of error include improper washing of the protein or having remaining cell debris after the incubation and centrifugation was completed. With contaminated Elutions, the Nanopure spectrophotometer measurements would be flawed. For characterization, sources of error included using the Aptamer stream buffer accidentally, pushing too hard into the wells and puncturing them, and heat shocking at the wrong time. The wells were slightly loose, which caused a slanted output after drying. The elution was 25-33% pure, which is low. Another band appeared near the protein, showing that contamination had occurred previously. The molecular weight is 25kDa. The samples were as follows: 1- whole cells before harvesting, 2- supernatant, 3- waste, 4- wash step, 5- Elution 1, 6- Elution 2. Without sterilization of the area and equipment, much more error could have occurred, perhaps causing more than the 2 proteins to appear on the gel.

__ Conclusion: __ A purple protein extracted from jellyfish (gbr22) was expressed in E. Coli bacteria, expressed and isolated, and then characterized through sodium dodecyl sulfate polacrylamide gel electrophoresis (SDS-PAGE). This process portrayed how proteins can be isolated using various wet lab techniques such as heat shocking, centrifuging, and incubating. Key processes were performed at a molecular level, such as the 6xHIS tag and Ni-NTA resin binding. This process of protein examination could be used further in Virtual Drug Screening to isolate proteins and test ligands on the molecules to see if the active sites bind readily with them.

__ References: __

1. Graslund, S.; Nordlund, P., Weigelt, J., et. al, Protein production and purification. //Nat Methods// 2008, **//5//** **(2), 135-46.**

2. Protein Expression and Purification Core Facility. [] (accessed April 15, 2013)