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__ Title: __ Novel approach to obtaining protein through bacterial host

__ Introduction: __ In many biochemistry and chemistry labs, one of the biggest parts in analyzing a specific protein is to somehow obtain significant amounts of it in a fairly pure form. However, a problem arises in that some proteins in certain organisms are only produced in minute amounts and the ways to gather the proteins directly from these organisms is near to impossible. With the advances of biotechnology, scientists have found a way to break these limitations by expressing and purifying proteins using different organisms as hosts. One of the most commonly used organisms for recombinant protein expressions is E. coli [1]. This host provides scientists a way to overexpress a protein for extensive study. E. coli is extremely versatile in the different types of proteins it can express. From bacterial and human proteins to protein complexes to human intermembrane protein [2]. If the protein of interest is naturally found in humans, then scientists can use E. coli to express the protein in large amounts without using actual human tissue. The method to express protein in these bacteria begins with obtaining a plasmid containing the target protein. The protein is usually fused with an affinity tag to enhance filtration and purity [2].

The goal of these protein labs was to overexpress and purify the gbr22 protein in E. Coli bacteria for future testing. Gbr22 is a fluorescent protein cloned from a coral from the Great Barrier Reef hence its name. The experiment was divided into 3 labs: protein expression, protein purification, and protein characterization. For the first lab, E. Coli bacterial cells were transformed with a plasmid containing the gbr22 protein. The cells were inoculated overnight and then frozen to express the recombinant DNA. For the protein purification lab, the cells walls of the bacteria containing the protein were destroyed to release the soluble protein, which was then filtered to obtain a pure elution. The purpose of the final lab was to test the success of the purification process. The protein samples were analyzed using gel electrophoresis to see the progress of the protein throughout the labs and to see if there were any other proteins present after purification. Through proper expression and purification, the gel should only display 1 protein band demonstrating a viable protein sample.

__ Materials & Methods: __

25µl of E. coli bacteria from New England BioLabs transformed with 1 µl of plasmid (pGEM-gbr22) encoded with gbr22 and an ampicillin resistance gene in a transformation tube. The solution was heat shocked, plated on an agar amp plate, and incubated overnight. On day 2, a sample from a single colony on the plate was transferred to 5ml of LB supplemented by 100µg/ml of ampicillin and incubated for hours. 0.625 ml of the starter culture was transferred to 25ml of LB with ampicillin of concentration 100µg/ml and placed in the shaking incubator for 24 hours. Bacteria was transferred to a 50ml conical tube and centrifuged for 10 minutes at 5000 rpm at 4 degrees celcius. 2.5ml of 1x PBS was added to the conical tube containing the pellet and vortexed (sample 1). .051 ml of stock lysozyme was added to the solution and vortexed again. The tube was placed in a -20 degrees celcius freezer.

1 µl of cyanase was added to the tube and then incubated at room temperature for 15 minutes. Solution was centrifuged for 20 min at 14000rpm at 4 degrees celcius in microcentrifuge tubes (sample 2). Supernatant was isolated and filtered using a .45 µm SFCA syringe filter, and .5ml of Ni-NTA resin/buffer mix was added to the filtered solution and incubated for 15 minutes. Solution was poured into a 20ml Bio-Rad chromatography econo column and flowed through into a waste tube (sample 3). It was run through next with 10ml solution of 1x PBS and 20mM imidazole (sample 4). Elution 1 and 2 were obtained by running 1x PBS and 250 mM imidazole through the column twice (sample 5 & 6). Using the nanodrop spectrophotometer, absorbance readings were taken at 280nm and 574nm.

Sample 1 was centrifuged for 5 minutes at 5000rpm and resuspended in 200µl of water. 40µl of loading buffer was added. 10µl of loading buffer were added to samples 2-6, and all samples were heat blocked. A protein standard (Pageruler prestained 26616 Thermoscientific) was added into well 1 of the gel. Samples 1-6 for the first partner were added to wells 2-7 respectively. Samples 4-6 from partner 2 were added to wells 8-10. After electrophoresis, imperial protein stain was added to the gel, and the gel was placed in the orbital shaker for 1 hour and washed 2 times after that. After 24 hours, the gel was dried at 75 degrees for 1.5 hours on a gradient cycle.

__ Results: __













__Calculations__

A = ** ε **bc .15 = (38880)(1)c c = .15/38880 c = 3.86 x 10^-6 M = .1mg/ml .1 mg/ml x 5.5ml = .55 mg yield





__ Discussion: __

From the gel nano drop readings and calculations, the concentration of the protein using the 280nm reading was 3.86 x 10^-6 while the concentration of protein using the 574 nm reading was 3.89 x 10^-6. These values are almost identical implying that the spectrometer was very precise. Our target protein was the one that is higher up on the gel based on the protein ladder and its molecular weight standard. The estimated MW of the protein from the gel was 28 kDa which is pretty similar to the actual MW of 25794.2 Da. On the gel, 2 protein bands formed on elutions 1 and 2 for the first implying that the solution was not pure. Since the intensities of the bands are equal, there is about 50% purity. However, for the second set of elutions, there is one very intense band and one very light band. This suggests that the purity of the protein solution is close to 100%, which means better yield.

The lyzozyme was used to break the cell walls of the bacteria to release the protein contained inside. Other enzymes such as Cyanase was added to digest the DNA/RNA and make the solution less viscous. HIS tags were encoded with the protein to enhance the filtration process. The Ni in the resin/buffer mix bound to the HIS tags on the gbr22 protein keeping it from flowing through the column along with the other cell components. Since gbr22 was supposed to be the only protein with 6 HIS tags, the resin/buffer could specifically bind to the target protein. Sample 1 contained the bacterial cells containing the protein. Sample 2 was the supernatant with the protein obtained after centrifuging. Sample 3 was the waste that flowed through the column. Sample 4 was the wash buffer containing cell components other than the protein. Sample 5 contained the pure protein, and sample 6 contained any left over protein. The difference between the wash and the elutions is that the elutions contained the protein. The elution buffer released the protein from the Ni-NTA mix.

A possible error that could have occurred would be incorrect amounts of solutions added to the bacterial cells in lab 1 which could have led to a very small number of cells not containing the gene. This would have reduced yield. Since the test plate did not have any bacteria on it, the error of contamination can be ruled out. Other errors that could have occurred could have been not centrifuging the compound enough so that there is still cell components in the solution. The resin also could have bound to other cell parts which would have affected the concentration determined by the nanodrop spectrometer. Finally, the biggest error in this experiment occurred in the final lab. The gel was punctured resulting in the samples mixing into each other's gels. Moreover, when the pipette was taken out of the gel, it pulled some of the sample out further misplacing the samples. This could have led to the 2 protein bands in the first set of elutions and the many dark regions above our target protein.

__ Conclusions: __

In this experiment, the protein gbr22 was expressed using a bacterial host E. coli. First, a plasmid containing gbr22 and ampicillin resistance gene was inserted into bacterial cells. The cells were incubated overnight to express the protein. Cell were then given additional amp to kill off any cells that did not accept the plasmid. The cells were then lysed open using lysozyme to release the protein within the cell. After isolating the protein solution, it was filtered through a column to purify the sample. Absorbance readings were obtained using the nanodrop spectrophotometer. To test the success of the purification process, protein samples from each step of the experiment were separated by electrophoresis. The first set of elutions showed that the purification process did not work very well. It produced 2 bands of equal intensity indication 2 proteins. The second set, however, validated the purification process with 1 band of very high intensity and another with almost no intensity. Therefore, future testing on the protein should use the second set of elutions. However, this experiment should be repeated to obtain a pure protein solution. This experiment can also be replicated for any protein that may be of interest for virtual drug screening. Expressing protein in large quantities makes it easy for many drug screening tests and binding tests.

__ References: __

[1] Baneyx, F. Recombinant protein expression in Escherichia coli. //Curr. Opin. Biotechnol.// **1999,** //10// (5): 411-21 [2] Protein production and purification. //Nat Methods.// **2008,** //5// (2): 135-46