EMILY+J

__Title: __ Expression, purification, and characterization of the gbr22 protein in E. coli bacteria.

__Introduction: __ The process by which a protein is expressed and purified varies depending on the protein, as different affinity tags, host cells, and strains will be best for each protein [1]. E. coli is a commonly used bacteria for expression due to the fact that a half of eubacteria and a tenth of eukarya proteins can be expressed in non-virulent strains [1] (Another reason that is implied is that it is competent!). Ampicillin as a selective marker is one strategy to ensure proper transformation, while a six-histidine affinity tag ensures proper purification [2]. In the subsequent lab, gbr22 protein will be expressed in E. coli bacteria and then purified, removing all other proteins from the sample. To confirm this was done correctly, a gel will be run to characterize the protein and determine its purity. The protein sample is expected to be pure and uncontaminated by any other proteins. (Great!)

__Materials & Methods: __ Given a gene inserted into an expression plasmid, the first step was to transform the bacteria with the plasmid. Using sterile technique, a control tube of E. coli bacteria and a DNA tube containing the plasmid and E. coli bacteria were prepared and incubated. An agar plate was made from each sample. After a night of incubation, a colony from the DNA plate was used to make a small culture, and from this culture a larger culture was grown. When this larger culture was purple in color, it was harvested and centrifuged. The spent media was decanted and the purple cell pellet saved. The cells were then re-suspended. Finally lysozyme was added to break down the cell walls of the E. coli bacteria. (Need to mention the name of the plasmid as well as the strain of the competent cells).

To purify the gbr22 protein, Benzonase was added to digest the DNA and RNA in the solution. Using the microcentrifuge, the supernatant, containing the protein, was separated from the insoluble material, and the debris pellet was discarded. A syringe filter was used to filter out large particulate matter prior to Ni-NTA affinity purification. During Ni-NTA affinity purification, a homogeneous mixture of Ni-NTA resin/buffer mix was added to the supernatant before pouring it into the column. An initial solution of PBS was allowed to flow through, dislodging matter stuck to the sides. The wash buffer was then used to remove proteins only loosely bound to the resin. The protein was finally released from the column with the elution buffer, which contained a higher concentration of imidazole. Using a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE), the absorbance was taken for both elution one and two at 280nm (n=2 trials). This step was then repeated at the maximal absorbance for the gbr22 protein, 574nm. The UV/VIS mode was used to take two readings of the absorbance in the visible range at both wavelengths. Using the output from the spectrophotometer and Beer’s Law, the yield of the protein was determined.

The samples collected were then used to determine the purity of the protein through an SDS-PAGE that separated the proteins by molecular weight. The samples were first prepared for the gel. The electrophoresis module was assembled by setting up the gel cassette and adding TGS buffer. After each lane was cleared, the MW standard was loaded into the first well, samples 1-6 and another set of samples 4-6 were loaded in the subsequent 9 wells, taking care not to puncture the gel. The gel was washed thrice, stain was added to the gel, then once dark bands were visible, the gel was washed twice more. The gel was dried and from the bands of intensity, the purity of the protein was determined.

__Results: __

Figure 1a: Agar containing ampicillin with no bacteria (BL21 (DE3)), and after a 24-hour incubation period at 37 degrees Celsius, no bacteria growth was seen. Spots seen in the image are merely condensation on the lid.

Figure 1b: Ampicillin positive agar plate containing both DNA (BL21 (DE3)) and plasmid DNA pGEM-gbr22, with bacteria cultures appearing as small yellow dots after a 24-hour incubation period at 37 degrees Celsius. Estimated over 100 colonies present.

Figure 1c: Ampicillin negative plate with no bacterial growth after an incubation period of 24 hours at 37 degrees Celsius. Predicted that bacteria from a cough would grow, but no growth was seen.

Figure 2: BL21 (DE3) bacterial culture that have successfully transformed with plasmid DNA pGEM-gbr22. This culture was obtained through growth from a single bacterial colony from the agar plate in Figure 1b.

Figure 3: Cell pellet after centrifuging the culture of BL21 (DE3) that had transformed plasmid DNA pGEM-gbr22 (as seen in Figure 2). Pellet weight is equal to 0.26 grams.

Figure 4: 5mL of the purified protein, Gbr22, after washing with 1X PBS and 250mM imidazole solution elution buffer. Elution 1 is purple in color.

Figure 5: 5mL of the purified protein, Gbr22, after washing with 1x PBS and 250mM imidazole solution elution buffer. Elution 2 is clear.



Figure 6: Run 1 of Elution 1, absorption measured to be 0.29 mg/ml by the Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) at wavelength of 280.

A=Ebc Absorption= 0.030 B; path length of the cuvette= 1mm Extinction coefficeient at 280 nm= 33850 /M*cm Molecular weight of gbr22= 25794.2 g/mol C=(0.30)/ (38850)= .1992 mg/mL

Yield= 0.1992 mg/mL x 5mL= **0.996 mg**

With the use of these same calculations, and with the appropriate absorbance reading and extinction coefficient at 574nm, the yield of the protein at it's maximal wavelength of 574nm was found to be 0.403 mg.

Figure 7: Image of gel, post drying. The MW standard is in the far left well, then samples 1-5 in the next 5 wells, and finally a different group of samples 4-6 in the final 3 wells. ( Need to be more specific with regards to what sample is in each well)



Figure 8: Molecular Weight standard for Pageruler Prestained Protein Ladder, Thermo scientific (product #26616).

__Discussion: __ <span style="font-family: 'Times New Roman',Times,serif;"> To ensure the initial uptake of the plasmid, antibiotic resistance was used as a selective marker. Ampicillin resistance, along with the gbr22 gene, was inserted into the expression plasmid, and when the bacterial colonies were grown on an Agar plate containing ampicillin, only the bacterial cells that successfully transformed the plasmid containing the protein of interest had the antibiotic resistance that allowed them to survive and grow.

<span style="font-family: 'Times New Roman',Times,serif;">Once these cultures were grown and incubated into a larger culture, the cells could be harvested. A sample of the colony was centrifuged and the pellet containing the bacterial cells was kept. Lysozyme was used to digest the cell walls of the E. coli bacteria and benzonase, a nuclease, reduced the viscosity by digesting both the bacterial DNA and RNA. This broke the proteins out of the bacterial cells, and thus began the purification process.

<span style="font-family: 'Times New Roman',Times,serif;"> In purification a six-histidine tag was used as an affinity tag on the protein of interest, which allowed the protein to bind to the nickel during Ni-NTA affinity purification. This caused the gbr22 protein to remain in the column while proteins originating from the bacteria, lacking the HIS tag and thus not bound to the nickel, were washed out. The wash buffer contained a lower concentration of imidazole, at 20mM, and removed the proteins loosely bound to the resin. The elution buffer contained 250 mM of imidazole, and at this higher concentration the imidazole competes with the histidine to bind with nickel, thereby removing the protein from the resin. <span style="color: #ff0000; font-family: 'Times New Roman',Times,serif;">(Great!)

<span style="font-family: 'Times New Roman',Times,serif;"> The samples used for the characterization gel were collected at various phases during the purification process. <span style="color: #ff0000; font-family: 'Times New Roman',Times,serif;">(And expression) <span style="font-family: 'Times New Roman',Times,serif;"> Sample one was from the original culture of bacterial cells, which were transformed with the plasmid containing DNA for the gbr22 protein and then grown into a large culture in the incubator. Sample two was a sample of the supernatant liquid, containing the protein, after centrifugation caused the insoluble fraction of the cells to settle into a pellet. Arising from Ni-NTA purification, sample 3 was the flow through from the initial PBS wash, sample 4 was that from the wash buffer and contains loosely bound proteins, sample 5 was that from elution 1 and contains the majority of the protein, and sample 6 was that from elution 2 and contains a minority of the protein.

<span style="font-family: 'Times New Roman',Times,serif;">From the gel, it was seen that the gbr22 protein had a molecular weight of 25 kDa based upon the molecular weight standard. From the literature in the protein purification lab, it was found that the molecular weight of gbr22 was 25794.2 g/mol, which is very similar to the results from the gel.

<span style="font-family: 'Times New Roman',Times,serif;">The wet cell pellet from the expression step weighed 0.26 g, but after protein purification, Nanodrop spectrophotometry determined the yield of the protein to be only 0.403 mg, using the maximal wavelength of 574nm. The nanodrop data showed the quantity of the protein, but a gel was run to determine the quality. The gel showed that there were multiple bands of intensity in the sample 5 well. For a pure protein, there should be only one band. As there were multiple, the protein is not pure, as these bands indicate other proteins with different molecular weights are present in the sample in addition to the gbr22 protein. Due to these contaminates, the protein is estimated to be 40% pure.

<span style="font-family: 'Times New Roman',Times,serif;">Sources of error could include unsterile lab techniques, thereby contaminating the samples with other bacteria, which would then express different proteins. Another error occurred during the stripping procedure of the column, but that did not affect the results of the lab. While the extra proteins that are contaminating the sample could have been a result of any contamination during the procedure, it is also possible that it occurred prior to that, when the gene was inserted into the plasmid. A second gene could have also been inserted by mistake into the plasmid <span style="color: #ff0000; font-family: 'Times New Roman',Times,serif;">(how so? isn't RE digest specific? add more, you're on the right track) <span style="font-family: 'Times New Roman',Times,serif;">, and thereby was able to be expressed in the bacterial cell, as it also contained the antibiotic selective marker. This possible error could be further explored if the gene in the plasmid is sequenced to ensure the insertion process did not introduce any new genes or mutations.

__<span style="font-family: 'Times New Roman',Times,serif;">Conclusions: __ <span style="font-family: 'Times New Roman',Times,serif;"> In the protein labs, a plasmid was transformed into bacterial DNA, and was subsequently overly expressed in a grown culture of E. coli cells. The bacterial cells were lysed, and the protein was purified, eliminating the insoluble cell fragments through centrifugation, and other proteins through Ni-NTA affinity purification. Finally, samples collected from the purification process were characterized in a gel that determined that the protein was not pure, as seen by multiple bands of intensity in the well containing the flow through from elution 1, which contained the most gbr22 protein. In the future, proteins will need to be expressed and purified before testing potential drugs for the target protein in the wet lab. However, these proteins will need to be pure to ensure accurate results for the testing of these ligands.

__<span style="font-family: 'Times New Roman',Times,serif;">References: __ [1] Nat Methods. **2008** Feb;5(2):135-46 [2] European Molecular Biology Laboratory. Protein Expression and Purification Core Facility. [] (<span style="font-family: 'Times New Roman',Times,serif;">Accessed April 16, 2013). [3] Thermo Scientific. PageRuler Prestained Protein Ladder 10-170K. [] (Accessed April 11, 2013).