ASHLEE+W

__ Title: __ Overexpressing bacterial proteins, Protein Purification, and Protein Characterization...the Steps Towards Discovering New Drugs

__ Introduction: __ When selecting a method of how to produce a recombinant questions (good sentence - awkward wording), many questions regarding strategic choices can be asked, such as: should the protein be expressed in bacteria; if bacterial expression is used, which strain(s) should be chosen; which good purification strategy should be used? [1] Protein purification is a series of processes intended to isolate a single type of protein from a complex mixture, which is crucial for the function, structure, and interaction of proteins. Chromatographic techniques, such as absorbance spectroscopy, and spectrofluorimetry, circular dichroism spectrometry can be used to study proteins in terms of their specific activity, stability, and molecular interactions [2]. When working in vitro, it is important to be able to purify a protein in isolation and signaling capability can be studied in detail. After purification is carried out, specific residues can be modified, allowing a protein to act as a catalyst. (good background information, but detailed background information relevant to your protein procedure as opposed to introducing others(spectrofluorimetry, circular dichroism) not used, may have been a better alternative) The purpose and objectives of these three labs, protein expression, purification, and characterization, were to ultimately overexpress a recombinant protein in bacteria by transforming and growing bacterial cells with a DNA plasmid in order to express the recombinant protein. These cells were preserved in order to break open the bacterial cells of the overexpressed protein in order to isolate and purify the protein, This isolated protein underwent electrophoresis to separate proteins and to make a gel sample of the gbr22 protein.


 * Hypothesis **

After completing the labs, protein expression, protein purification, and protein characterization, recombinant protein will be expressed, and it should be purified using a nickel column.

__ Materials & Methods: __ In order to ensure safety in the lab, Personal Protective Equipment, PPE, was utilized and worn at all times, including a lab coat, gloves, and safety goggles. The bacterial waste must be treated with bleach for twenty minutes and then dumped into the proper waste container to be removed. The first steps to overexpressing a protein in bacteria are cloning the gene and inserting the gene into an expression plasmid. The presence of the gene is then confirmed and the gene is sequenced to make sure mutations have not occurred. There were three plates: a experimental plate with DNA, a control plate that has no DNA and colonies to grow without the help of plasmid, and one plate for “fun.” 25 microliters of bacteria was added, spun down using a mini-centrifuge, and 1 microliter of plasmid was added to the bacteria in the DNA tube only. There was a heat shock used in the incubator, which was used to transform the expression strain of bacteria (//E.coli// BL21(DE3)) with the plasmid. The bacteria were then grown on an LB agar plate with antibiotic to select for colonies that had the gene for antibiotic resistance. When the bacteria were grown on the LB agar plate or in LB broth containing ampicillin, only those bacteria that have been transformed with the plasmid containing the ampicillin resistance gene survived. After modifying the overexpressed purple protein (gbr22) in protein expression, in protein purification, the protein was modified to have six histidine residues added to the C-terminus, which could be easily used to separate the protein from other cellular proteins. (this sentence has some grammatical errors - the protein was not modified in the purificaiton, your protein already had the transcribed and translated his tag in its DNA before expression) The histidine residues binded to cations, such as nickel, which could be immobilized on a column matrix such as Ni-NTA agarose ( form a new thought/sentence here because there is ambiguity in your understanding of the His tag purification - imidazole would compete for the binding and cause the release of the protein after being immobilized) by adding imidazole that competed with histidine residues for metal binding. The E.coli cells that expressed the purple protein were lysed open, the lysate was clarified by centrifugation, the protein was purified using Ni-NTA affinity chromatography. The Nanodrop spectrophotometer was then used to measure the concentration of the protein used. Samples were collected to be analyzed for protein characterization using gel electrophoresis and UV-Vis spectroscopy. The technique used to follow the expression and purification labs was the characterization of the purple protein, gbr22. The six samples collected in the previous lab were mixed with dye and then heated (heated why?). Before gel electrophoresis, the samples were injected into the lanes of the gel, using the protein ladder as a reference for comparison. After letting the gel rinse overnight, the gel was then dried and analyzed in order to estimate the concentration of the protein solution, pgbr-22 (the gel is more of an analysis of purity, than concentration).

__ Results: __

Figure 1a: BL21 (DE3) bacteria transformed with pGEM-gbr22 plasmid with LB+Amp media grown at 37 degrees Celsius.

Figure 1b: BL21 (DE3) bacteria with LB+Amp media grown at 37 degrees Celsius.

__ : __ Figure 1c: Plate that contains bacteria grown from swabbing two students' cell phones with LB media grown at 37 degrees Celsius. Figure 2: BL21 (DE3), pGEM-gbr22 Purple Culture (with DNA) in the evening on Day 3 after spending 24 hours in the shaking incubator at 37˚C and 250 rpm.

Figure 3: BL21 (DE3), pGEM-gbr22 Purple Culture cell pellet (with DNA) in the evening on Day 3 after centrifuging for 10 minutes at 4°C and 5,000 rpm. Weight of 0.42 g.



Figure 4: Run 1 of Elution 1, the absorption measured to be 0.29 mg/ml by the Nanodrop Spectrophotometer (Thermo Scientific, Wilmington, DE) at a wavelength of 280 nm.

Maximal Wavelength: A=EBC, C=A/EB A= 0.030(10)=0.30 0.30= (38,850(1)(c)) c= 7.72x10^-6

7.72x10^-6 mol (25794.2 g/mol) = 0.1992 mg/ml (general wavelength for all proteins at 280 nm)

0.1992 mg/ml x 5ml= 0.996 mg

With the same calculation, the absorbance at the maximum wavelength of the purple protein at 574 nm was 0.403 mg.

Figure 7: The final dried gel run during protein characterization. The stained bands represent proteins, while the intensity of each band shows the purity of each protein. The ladder is displayed in lane 1, along with samples 1-6 displayed in lanes 2-7.

Figure 8: Molecular weight standard for the protein used during protein characterization(What is the brand of the ladder used? - Thermoscientific) .

__Discussions__:

Lysozyme was used to break down the cell wall of E.coli, thus isolating the protein further adding benzonase/cyanase, which is used to break down the DNA and RNA. this resulted in the purification of the protein. During this process, a six-histidine tag was used as an affinity tag to allow the protein to bind to nickel during this Ni-NTA affinity purification. Bacteria, lacking the HIS tag, were not held in place, and were washed out. Sample 1 was from the original culture of bacterial cells transformed for the gbr22 protein. Sample 2 consisted of the protein in the liquid, after centrifugation, which resulted in settling into a pellet. Sample 3 was composed of the original PBS wash buffer, which contained a lower level of imidazole. Sample 4 contains loosely bound proteins in the wash buffer. Sample 5 and 6 were form elutions, more concentrated than wash buffers, 1 and 2, but elution 1 was more concentrated with proteins.

During this lab, using electrophoresis, the samples prepared in the previous two labs, expression and purification, were used to be analyzed. Using the results, the molecular weight of the gbr22 protein was estimated, which was somewhere between 15 and 25 kilodaltons. Because the samples were diluted, and in an attempt to maintain a good gel, modifications were made, such as the times the gel was rinsed, the amount injected into each lane of the gel differed, and the amount of time it was left in the staining solution. It was determined that the proteins were in the same area as the online data that it was compared to. The differences were in the intensity of the bands, which were because of the dilute solutions. There are two protein bands instead of one because one of the bands is some sort of contamination. The contamination is from the original pgbr22 plasmid that was transformed. In every experiment, there are sources of error that can be prevented in order to ensure accuracy and precision. Sources of error could stem from not making sure that the incubator is turned on and working properly, leaving a loose flask while it is shaking, esuring that the shaking incubator is operating at the correct setting, making sure that the amounts of solution collected are correct, keeping track of one's solutions, and by paying attention to one's technique when injecting protein solutions into a gel are all sources of error that can easily be prevented in order to prevent contamination in one's results. (Talk about your results in the discusion e.g. quantity of protein expressed, how successful your purification was based on the number of bands etc.)

__Conclusions:__

After using electrophoresis, the samples prepared in the previous two labs, expression and purification, were used to be analyzed. Sodium dodecyl sulfate polychylamide was used to separate proteins in the sample (SDS-PAGE). The molecular weight of the gbr22 protein was estimated. The concentration of the protein solution was estimated using the correlation of the data and UV-VIS spectroscopy measurements that were made in the last lab. These labs were beneficial in the sense that the new knowledge obtained over protein characterization can be utilized to express proteins and enzyme assays involving testing drugs as the proper technique and ability to analyze data are becoming understood.

__ References: __ __ [1 __] Graslund, S.; Nordlund, P., Weigelt, J., et. al, Protein production and purification. Nat Methods 2008, 5(2), 135-46. [2] IBMC. Protein Production Purification Unit. (accessed April 2013).