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__ Title: __ Expression, Purification, and Characterizationof gbr22 Fluorescent Protein __ Introduction: __ Using __#|recombinant proteins__ and isolating proteins through protein purification has been popular in recent years and is used frequently. [1] To gain protein understanding, the process of cloning, purification, and characterization are one of the most successful methods that can be used. [2] Proteins of interest are from a natural source and they must be first extracted from this source, cloned into vectors, and then expressed in a host such as E.coli. Then the protein goes through a three __#|step__ process: expression, purification, and characterization. The protein used for this lab was gbr22, and through this lab it was extracted using centrifugation, purification, and characterization from doing gel electrophoresis. [1] The objective of this lab was to isolate a protein, and this was done in a 3-step process. The first was to over express the __#|recombinant protein__ gbr22, then purify it using chromatography, and then characterize the protein of interest with an SDS-PAGE gel. The hypothesis is that the end result is a sample of the gbr22 that is purely the target protein, and the gel from SDS-PAGE will only shown one protein band.

__ Materials & Methods: __

E.// coli // BL21(DE3) bacterial cells (New England Biolabs, Ipswich, MA) were transformed with the pGEM-gbr22 plasmid. Through centrifugation the plasmid was inserted into the host cells (25 uL), and they were put on ice and then heat shocked. After that they were put on ice for 2 min and then 200 uL of SOC media was added and put in the shaking incubator for 30 min. Then bacteria/SOC mixture (50 uL) was grown on one control plate and one LB-agar and ampicillin plate and incubated overnight. A starter culture was grown in LB, ampicillin and one transformed bacterial colony and incubated for 8 hours. Culture plates were stored in the 4C fridge. 25 mL of the fresh LB, 50 uL of ampillicin, and 0.625 mL of the culture were transferred to a sterile 125 mL flask and put into the shaking incubator for 16-24 hours.

2 uL of benzonase was added to the thawed transformed cells. Lysate was centrifuged and the supernatant was saved. The supernatant containing the protein was filtered through a .45nm syringed filter and run through a column with .5 ml of Ni-NTA resin/buffer. Three samples were collected and then wash elution was added and this was sample 4. Then elution was added twice and samples 5 and 6 were collected. Nanodrop spectrophotometer was then used.

The __#|last__ step was to load the samples (prepared with 6x loading buffer) in gel wells, and they were ran for 25 minutes. Gel was rinsed multiple times and then stained with Imperial protein stain, rinsed, and then dried on the Gradient cycle.

__ Results: __

Figure 1: Agar- amp plate containing E. coli colonies inserted with pGEM-gbr22 plasmids.

Figure 2: Control plate, contains no amp resistance and no DNA and thus no living bacterial colonies

Figure 3: Fun plate on agar plate, bacterial colonies from human saliva. Colonies of bacteria are visible



Figure 4: Wet pellet acquired from centrifugation of bacteria culture transformed with pGEM-gbr22 plasmid DNA (.32 g)

Figure 5: Elution 1 and 2 of the pGEM-gbr22 protein

Figure 6: Flask containing culture of bacterial cells transformed with plasmid DNA pGEM-gbr22.

Figure 7: Absorbance spectrum for Elution 1 at 280 nm (.25mg/ml) using Nanodrop spectrophotometer.

A = ** ε **bc .25 = (38880)(1)cc = .25/38880c = 6.43 x 10^-6 M 6.43 x 10^-6 M x (25,794.2 g/mol ) = .165858 mg/ml .165858 mg/ml x (5 ml) = .8293 mg



Figure 8: Final gel after being dried for 1.5 hours. Total of 10 wells each from different stages of lab.

__ Discussion: __ This lab allowed for the isolation of the protein of interest, gbr22. After competent E.coli cells were successfully transformed with the pGEM-gbr22 plasmid, bacteria grew in colonies in an LB+amp agar plate. The cells with the plasmid are able to grow on these plates because they contain a specific gene for ampicillin resistance. As a result, the cells with the protein of interest can be easily identified from the rest of the cells.

After the gbr22 was overexpressed, lysozyme broke the cell walls of the bacteria so that proteins could be released (purification step). Ni-NTA chromatography was used to separate and purify the gbr22 protein. Benzonase was also used to digest DNA and RNA, which could be a source of error. Since gbr22 protein is modified with 6 HIS residues at the C-terminus, the HIS affinity to Ni-NTA allowed the protein to stay on, while other proteins simply flowed through. The wash buffer containing imidazole competes with the HIS residue and therefore with a high concentration of imidazole (elution buffer), the gbr22 protein was released.

The SDS-PAGE gel had wells with each of the 6 samples. Sample 1 contained the bacterial cells containing the protein, sample 2 was the supernatant after centrifuging, sample 3 was the waste that flowed through, sample 4 was the wash buffer, sample 5 contained pure protein, and sample 6 contained any left over protein. From the dried gel, it could be seen that there were 2 protein bands formed on elutions 1 and 2, which implies that there was only about 50% purity. However, for the second set of elutions the second band was only faint so it could be inferred that this was closer to 100% purity, which also means a better yield. The estimated MW of the protein by looking at the molecular weight standard is about 23 kDa, which is close to the actual MW of 25794.2 Da.

Some sources of error could definitely be human error such as slightly wrong amounts of solutions were added into the bacteria or the wrong amount of imidazole was put into the solution. In addition to this, the two bands that were present in the final dry gel indicates that there must have been impurities in the lab in some point in time. This means that there was another protein other than the protein of interest (gbr22) that was overexpressed and characterized. However, the biggest error that took place was during the third part of the lab. The gel was punctured with a pipette and therefore some of the samples mixed together and some leaked out. __ Conclusions: __

Through this lab, the three steps of protein isolations was carried out. First E.coli cells were successfully transformed with the pGEM-gbr22 plasmid. Then these cells were then purified using Ni-NTA affinity chromatography, and finally the protein was characterized with SDS-PAGE analysis. The Nanodrop spectrophotometer was used to find the amount of protein, and then the SDS-PAGE was used to predict the molecular weight of the protein. The final dry gel showed that there was a purity of 50%, and the estimated molecular weight for gbr22 was 23 kDa. The techniques learned in this lab can be used in the future for VDS labs in which proteins need to be identified. Using these techniques, a protein can be isolated, expressed, purified, and characterized so that the protein of interest can be studied.

__ References: __ [1] Acton, T.B.; Albeck, S.; Almo, S. C.; Anderson, S.; Arrowsmith, C.; Atwell, S., Protein production and purification. Nat Methods 2008, 5, (2), 135. [2] Baneyx, F. Recombinant protein expression in Escherichia coli. // Curr. Opin. Biotechnol. // ** 1999, ** // 10 // (5): 411-21