MIGUEL+T

__ Title: __ Perks of VDS: Expressing, Purifying, and Characterizing

__ Introduction: __ There are many techniques to produce and purify proteins, therefore, protein production and purification is a process that has been approached through many angles (this is redundant). Usually, the final goal is to be as accurate and precise as possible, while saving both time and money. This doesn't come easy. Some techniques used for this include structural genomics and cloning. "The most common methods now used in our groups to clone target genes into the requisite expression vector rely on homology-based approaches, using either recombination enzymes[|25] or ligation-independent cloning (LIC)[|26]." [1] In these particular labs, proteins were purified, expressed, then characterized. When one purifies a protein, he/she attempts to extract the protein from a certain mixture in which the protein resides. When a protein is expressed, the results are used to denote the measurement of the presence and abundance of one or more proteins in a particular cell or tissue. When a protein is characterized, it is studied by most of its aspects. Therefore, the main objective of this lab was to clean, isolate, and study a protein. In this case, that protein was pGEM-gbr22. These three procedures were achieved through 3 different labs, and all of them took several days to do. Like mentioned before (Grammar), all three of these processes can be achieved through different ways, and that is all up to the person doing the lab to decide. (Each way can benefit a certain for better or for worse). Once all of this was completed, data was analyzed and measured. If all steps were followed correctly, and human error kept to a minimum, then the protein could successfully be expressed, purified, and characterized. (either describe the steps in a bit more detail. you haven't talked about how exactly it was done in this lab)

__ Materials and methods: __ These three labs included multiple steps each, but the main ones shall be mentioned here. For the expression lab, the steps were done over 3 or 4 days. The purpose of the M&M is to summarize the procedures. you don't need to write that first sentence. The first major step in the expression lab was an overnight transformation. This included a plate with microcentrifuged DNA plasmid. Then, after a few minor steps, such as heat shocking and adding SOC media, the DNA was then added to the plates with colirollers. The next day consisted of two halves. Ideally, one should be done in the morning and the other in the afternoon. In the first half, a culture was grown in LB supplemented with Ampicillin. During the second half, the culture was made larger by adding more ampicillin and LB. The flasks were then left in the incubator overnight (16-24hrs). Day 3 consisted of harvesting the cells of the protein. The specimens were centrifuged again, and bacterial pellets were obtained. The pellets were then resuspended in solution. The second lab consisted of purifying the protein. In this lab, buffers were used to purify the proteins which were overexpressed the week before. Several enzymes such as cyanase and lysate were added to help break down the outer coatings of the proteins. Only the pure, naked protein was wanted. The sample was then centrifuged yet again, and once all of the minor steps were done, a spectrophotometer called Nanodrop was used to measure at maximal wavelength. The third lab consisted of characterizing a protein. This lab was more complicated than the last two, and included using gel electrophoresis to analyze the samples used in the previous two labs. Materials yielded in the purification lab were used in this lab, and were key to the starting process. After all solutions had been made ,electrophoresis was used to separate the protein and give it a negative charge. After all of these steps, a gel was obtained which was used to analyze the protein. Before that could happen, though, it was placed in a dryer. A couple of hours of drying later, the gel was ready to be analyzed. Don't forget to say why you did each step. __ Results: __

Figure 1- Positive control plate of DNA with BL21, the bacterial cell with pGEM-gbr22, which is the plasmid of the transformation exhibited after a 24 hour incubation period at 37 degrees Celsius.

Figure 2: Negative control plate with BL21 (DE3) bacterial cell and no plasmid.

Figure 3: "Fun Plate"- contained mouth bacteria, no plasmid



Figure 4: The culture of 25 ml LB, 50 ul ampicillin, BL21(DE3) bacteria and pGEM-gbr22 plasmid after being left for 16-24 hours in the shaking incubator in a 50 ml conical tube.

Figure 5: The BL21(DE3) with plasmid pGEM-gbr22, LB, and ampicillin pellet after being centrifuged for 10 minutes at 4 degrees Celsius and at 5,000 RPM. It weighed 0.34 grams.

Figure 6: Elution 1, including buffer obtained with purified protein gbr22. Figure 7: Elution 2, containing buffer obtained with purified protein gbr22.

Figure 8: Absorbance vs. wavelength reading at a wavelength of 280 nm for protein gbr22 on trial 1 (n=2).



Figure 9: Molecular Weight Standard (Thermo Scientific PageRuler Prestained Protein Ladder; 4-20% Tris-glycine gel (SDS)-PAGE; Product #26616) Figure 10: Destained gel after 24 hours on orbital shaker

Figure 11: Dried gel after an hour and a half in the gel dryer. Please label the wells.

Beer's Law Calculations:

Beer's Law: A = Ebc Molecular weight of gbr-22 = 25,794.2 g/mol 280 nm A = 0.33, E = 38,850 L/mol*cm, b = 1 cm  c = A/Eb = (0.33)/(38350 L/mol*cm)(1 cm) = (8.57 x 10-6 mol/L)(25794.2 g/mol)= 0.221 mg/ml concentration Yield = cV = (5ml)(0.221 mg/ml) = 1.105 mg yield

__Discussion:__ Throughout these three labs, as shown above, proteins were expressed, purified, and characterized. Enzymes such as Benzonase and Cyanase were used to help break down and purify the mixture that the protein was in. Many tedious, extraneous steps were done throughout these labs. One of these steps included using HIS tag, which, for purification process, the HIS tag works by the protein that has been made so that it has 6 histidine residues added to the C-terminus, which is used to separate that particular proteins. These residues will bind to the nickel that is immobilized on a column matrix such as Ni-NTA resin. Six samples were obtained fro the first two labs, and each sample contained a different substance in it. The first sample was obtained from the E.Coli cells that are present with proteins and other such debris. The second sample contained a soluble fraction after the lysozyme was added. Sample 3 had the Ni-NTA resin flow through the column after the syringe filtering where the sample was collected after letting the protein go through and contained proteins that did not in fact stick to the nickel. Sample 4 came after the wash solution was run through and contained proteins loosely stuck to the Ni-NTA resin. Sample 5 was collected after Elution 1 buffer was run through where it contained the purple protein and had one dark band indicating the purple protein, which is where only 1 band should be present. Sample 6 was collected after the elution 1 buffer, which becae elution 2 as it was used a second time and extra imidazole was added. It is estimaded that the purity of our final sample was about 35%. __ Conclusions: __ ​In these three labs, the protein pGEM-gbr22 was expressed, purified, and characterized. In the first part of the lab, bacterial cells were transformed with the plasmid (pGEM-br22), which overexpressed the purple protein. In the second lab, the protein was extracted from the bacterial cell with the help of a few enzymes. This eased the purification process. Then, the protein was purified even more by the affinity tag &Ni-NTA resin where centrifugation release the insoluble cell debris after. This all led to the electrophoresis process which helped characterize the protein. You should put this in your intro. this is the purpose of the lab. it's worth saying twice All of these steps lead to further the experience of the student in the VDS process. Even though the protein was not even close to being 100% purified, it yields crucial experience and knowledge. All of this well help with future implications of VDS, and will hopefully help in the research for the discovery of new drugs/diseases. How is it effective outside VDS?

__ References: __

1. Acton, T.; Albeck, S.; Almo, S.; Anderson, S.; Arrowsmith, C.; Atwell, S., Protein production and purification. //Nature publishing group.// **2008**. (2), 135-46.

2. Protein production and purification.Nat Methods. 2008 Feb; 5(2):135-46.