JAMES+T

__ Title ____ : __ The Color Purple of pGEM-gbr22 protein expressed in BL21(DE3) //E. coli// bacteria

__ Introduction: __ Acquiring large quantities of pure protein in an efficient and cost effective manner is difficult. Recent progression via commercially available systems has made technology to produce proteins more common [1]. Proteins can be made in a variety of mediums, including bacteria, yeast, and even insect or human cells [1]. After being purified of other debris used in expression, these proteins can be used for study in non-pathogenic, stable environments that are more easily and safely monitored than in vitro [1] [2]. For this experiment, pGEM-gbr22 will be introduced to BL21(DE3) //E. coli// cells and overexpressed. The protein will be purified of everything else and its absorbance will be measured. To verify successful expression of the protein, the protein will be run through electrophoresis. The purified protein will be purple in color and will appear at the corresponding position in the gel for the protein after electrophoresis. __ Materials & Methods: __ //E. coli// cells (BL21(DE3)) were introduced to and heat shocked with plasmid (pGEM-gbr22). This mixture was incubated in an Aar plate with ampicillin. These cultures were grown overnight with the plasmid; two colonies of the bacteria were added to LB media with ampicillin. The bacteria, in growth phase, were set in a shaking incubator at 37 degrees Celsius and left for 24 hours. A 50 ul sample was taken and the now purple mixture was centrifuged and the protein was harvested. Lysozyme and cyanase were added to the mixture to isolate the protein. After enough time, the lysate was centrifuged and a sample of supernatant was taken. The supernatant only was extracted and syringe filtered. The protein was bound to Ni-NTA resin/buffer mix and run through a Bio-Rad chromatography Econo column with water, pre-made 20 mM imidazole wash buffer, and twice with pre-made 250 mM elution. A 50 ul sample of each of these were taken after being put through the column. The 6 samples (lysate, soluble supernatant, flow-through, wash, elution 1, and elution 2) were placed in different cells on an SDS-PAGE gel after being prepared for electrophoresis. This was run for 25 minutes. After staining and de-staining, the gel was dried and analyzed. Elutions 1 and 2 were individually run through the Nanodrop® spectrophotometer (Thermo Scientific, Wilmington, DE) to determine absorbency. __ Results: __





Calculations for the concentration at 280 nm: A=Ebc A = 0.479mgml -1, E = 38850 M -1 cm -1 , b = 1 cm 0.479 = 38850 M-1cm-1 * (1cm) * c  0.0000123 M

Calculations for the yield of pGEM-gbr22 protein: c*MW = X mgml -1 X mgml -1 * V = yield c = 0.0000123M, MW = 25794.2gmol -1, V = 5ml 0.0000123M * 25794.2gmol -1 = 0.317 mgml -1 0.317 mgml -1 * 5ml = 1.58 mg of pGEM-gbr22 protein

__ Discussion: __ After introducing the pGEM-gbr22 plasmid vector to the BL21(DE3) bacterial cells, colonies should have grown in the Agar plate containing the BL21(DE3) competent cells with the plasmid. As shown by figure 1a and figure1b, only the bacterial cells with the plasmid vector grew. Two of these colonies were scraped off the Agar gel and incubated in LB media and ampicillin to express the protein in a larger culture. As shown by figure 2, the protein was expressed as the culture had turned purple in color. The protein was then purified. The culture was centrifuged; the supernatant was removed as shown by figure 3. Lyozyme and cyanase were introduced to the mixture in order to break down the cell walls and digest the DNA/RNA of the bacteria respectively. The lysate mixture was centrifuged again and the supernatant containing the protein was extracted. After syringe filtering, this was mixed with Ni-NTA resin/buffer mix. HIS tags on the pGEM-gbr22 protein stick to the large nickel bead in the Ni-NTA resin/buffer mix. Other debris and protein that were not stuck to the nickel bead would flow through the column and be washed away with the 20mM imidazole wash. The stronger 250 mM imidazole buffer was then used to elute the protein of the Nickel bead. Given that the elution 1 was purple, purple protein had been successfully stripped off the nickel and was no longer in the column. After adding more elution buffer, the mixture was only clear and without protein as shown in figure 4. Samples from the elution 1 with the protein were put through a spectrophotometer to record the absorbency at 280 nm. Using Beer’s law, the concentration could be determined and used to calculate the yield for the protein. Six samples taken throughout the experiment were put through electrophoresis along with a protein ladder in order to confirm the expression of the protein. Elution 1, sample 5, was in lane 6 of the gel shown in figure 6. The protein, when compared to the protein ladder, appears to weigh between 25 and 35 kDa as shown by figure 6 and 7; the molecular weight of pGEM-gbr22 is in that range (25794.2 gmol-1). One major source of error in this experiment was the failure of the first electrophoresis module fully loading the gel. The gel had to be switched into another module causing leakage of samples. As such, the purity of the protein in lane 6 appears to be around 35%. Other sources of error include ill preparation of chemicals, improper calculation, possible faulty equipment, and contamination due to failure to use sterile technique; all of which would affect how much protein was expressed and skew the final yield calculation. __ Conclusions: __ After introducing competent BL21(DE3) //E. coli// bacteria to the plasmid for purple protein (pGEM-gbr22) and incubating, the plasmid was taken into the bacteria and successfully overexpressed. The purple bacteria containing the protein were then broken down and the protein was purified using nickel and elution buffer. The absorbency of the protein was measured via spectrophotometry. Samples taken during expression and purification were put through electrophoresis to confirm that the protein had been expressed. This was confirmed as the elution 1 was purple, and the protein band in lane 6 of the gel was located between the marker for 25 kDa and 35 kDa. The absorbency was used with Beer’s law to calculate the concentration of the protein, which then was used to calculate the yield: ~1.58 mg of pGEM-gbr22 protein. The methods used during this experiment will be used in future labs to express more protein and verify that the correct protein has been expressed. Once expressed, these proteins can be used, for example, to test for inhibition when introduced to certain potential inhibitors. (good job) __ References: __ [1]. Nat Methods. **2008** Feb; 5 (2): 135-46. Protein production and purification. [2]. Protein Expression Questions. http://www.molecularsciences.org/book/export/html/49 (accessed April 16).