Max+M

__ Title: __ Characterization of pGEM-gbr22 protein expressed by recombinant //E//scherichia//. Coli//

__ Introduction: __ Protein production and purification can be used to investigate many properties of a protein. [1] The studying of proteins can lead to discoveries that could have therapeutic uses or to just broaden microbiological knowledge. The study of a particular protein and its characteristics can be a difficult task to initiate because there are so many methods that can be implemented into the studying of proteins. [2] In this lab we became familiar with the most common methods for expression a recombinant protein and purifying the protein for further analysis. Recombination-based methods were implemented by inserting a long chain of repeating Histidine amino acids at either end of the protein sequence. [2] This sequence is vital for the tracking of the protein in later procedures and to validate that the target protein was expressed and purified. In this experiment, we set out to express a protein purple in color from a coral located in the Great Barrier Reef. This florescent protein was hoped to be successfully expressed from the plasmid pGEM-gbr22. The purple color was used to validate the presence of the protein. From this expression we hoped to find its molecular weight range from gel electrophoresis and to conclude whether the correct protein was captured using Nanodrop Spectrophotometer data. If performed correctly. This experiment would lead us to our target protein and widen our understanding of protein expression and purification.

__ Materials & Methods: __ Our plasmid’s host, //E.coli// BL21, carried a gene for ampicilian resistance; therefore the transforming of the competent bacterial cells, provided by New England Biolabs 240county Road Ipswich, MA 01938-2723 **,**was performed on Lb agar AMP. Following a 45 second heat shock and the addition of SOC media to prevent contamination, the plates were incubated at 37⁰C overnight. The next morning the starter culture was grown in LB supplemented with 100 µg/ml ampicilian. A single colony growing on the LB agar Amp plates were then transferred into each of the two culture tubes containing 5 ml of LB and cultivated for 8 hours at 37⁰C. After the hours passed, 0.625 ml of the starter culture was transferred into a 100µg/ml LB/ampicilian solution and incubated over night at 37⁰C. Then, since our culture was already purple, we collected a 500ul sample of the culture. 50ml of the culture were then centrifuged at 5000rpm for 10 minutes to form a pellet of cells. Then in a 1xPBS working solution, 50µg/ml of stock lysozyme was added to form a 1mg/ml solution. The next step was to allow the lysing of the cells by the lysozyme and the adding of Bensonase, and a 50µl sample of this supernatant was taken after centrifuging. A wash buffer of 10 ml of final concentration 1xPBS with 20 mM imidazole and an elution buffer of 10 ml of final concentration 1xPBS 250 mM imidazole were created and used in the filtration steps for samples 3 through 6 following the addition of 0.5 ml of Ni-NTA for the purification of the supernatant solution. UV-Vis spectroscopy was then used to estimate the concentration of our final purified protein, found in “elution 1.” Protein characterization performed by the addition of 100ml of 5XTGS buffer to make a 1xTGS buffer into sample one and 10ul of 6x sample loading buffer into each of the other samples. 7µl of MW standard, (Prestained Protein Ladder #SM0671) provided by Fermentas, Page Ruler ™, was added into the second well of the Gel cassette and 20 ul of each other sample into the remaining wells in the Gel cassette. Then turn on for 25 minutes at a voltage of 200V. Wash with Imperial protein stain for 1.5 hours and then with nanopure water over night. The next day, the Gel was dried in the Biotech Lab. A Picture was taken prior to the drying just in case the Gel did not survive the drying cycle. Then the Gel was dried.

__ Results: __ Image 1 Image 1: Incubation period= 24 hours at 37⁰C. There is no substantial bacterial growth is present due to the presence of ampicillin.

Image 2

Image 2: Incubation period= 24 hours at 37⁰C. There is a substantial amount of //E. coli// BL 21 which expresses the pGEM-grb22 gene and is resistant to ampicillin. The antibiotic killed of any bacteria that did not contain the resistant plasmid.

Image 3



Image 3:Control plate (fun) into which a person sneezed. No ampicillin present and was treated as a test to see if any abnormal growth of microbial organisms would be present.

Image 4



Image 4: //E. coli// BL21 bacteria that expressed the pGEM-gbr22 protein in a 25 ml solution of LB broth and ampicillin. Incubated for 24 hours at 37⁰C on a shaking water bath incubator.

Image 5



Image 5:Centrifuged pellet containing //E. coli// BL21 cells that expressed the pGEM-grb22 protein using Allegra X-15 bench top centrifuge. Pellet weight= 0.65 grams Image 6a

Image 6a:Elution 1 contains the great majority of the pGEM-grb22 protein as it was washed with a 5 ml solution of Ni-NTA.

 Image 6b



Image 6b: Elution 2, washed with the same solution, was used to catch the remaining protein that was not released while collected Elution 1 Image 7

Image 7: Nanodrop emission spectra for 2μl from Elution 1 containing pGEM-grb22 taken at a wavelength of 280nm. Beer's law {absorbance= ebc where e is the molar absorbtivity (units 1/Mcm), b is the path length, and c is the concentration} could be applied with A=0.54, e=1, b=38850 and c is the unkown to where a final concentration of 0.13785mg/ml was calculated.

Image 8 

Image 8: Nanodrop emission spectra for 2μl from Elution 1 containing pGEM-grb22 taken at maximal wavelength of 574nm. Beer's law was also applied to this data but this time A=0.8, e=1, b=118300 and c was still the unknown to where a final concentration of17.44 mg/ml was calculated. This showcases that a higher concentration was present at the maximal wavelength that at a wavelength of 280nm. Image 9

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Image 9: Gel electrophoresis at 200V for 25 minutes prior to drying. Furthest to the left is the standard ladder comparison and is supplemented by the image alongside the image of the gel. The next nine columns are the results of samples 1 through 6 from one person’s procedures and 4 through 6 of another. The second to last column shows that our protein was expressed with relative purity as there are not lines of equal intensity. This line also allows the estimation of the 26 kDa molecular weight for the protein. Actual molecular weight of protein= 25794.2 kDa

__ Discussion: __ <span style="font-family: 'Calibri','sans-serif'; font-size: 14.6667px;">Due to the florescent purple color of the protein we were expressing, it was evident whether the protein was present or not, but in order to isolate our protein and to eliminate much of the waste produced by the lysosyme as it broke down the plasma membrane in addition to the waste produced by Benzonase as it broke down the DNA and RNA molecules within the cells. There were six different samples taken throughout the procedure. Sample one had cells intact, sample two had lysozyme and Benzonase added, sample three was taken after the addition of Ni-NTA which was used to bind to the Histidine tail located at the end of the of the protein of the protein sequence, sample four was the results of a wash that was performed to get proteins only loosely attached to the resin, and samples 5 and 6 were taken from elution one and two which were used to release the protein from the Ni-NTA by using concentrated imidazole. Recall that the Elution was of a much higher imidazole concentration than the Wash solution. The His tag system works by giving a location for which the Ni-NTA to binds to and perform a better job at isolating our target gbr22 protein. Based off Gel analysis, gbr22’s estimated molecular weight was about 26 kDa, and this corresponds with the actual molecular found before. Since there was no other band of equal intensity within sample five on the gel, the protein was about 90% pure. Sources of error were relatively clear to find within this lab because they consisted of poor aseptic techniques while dealing with the bacterial and wrapping the gel in saran wrap that was thicker than what should have been used. This thick saran wrap resulted in the gel not surviving the drying cycle.

__ Conclusions: __ <span style="font-family: 'Calibri','sans-serif'; font-size: 14.6667px;">This lab was performed to determine whether we had the ability to over express, purify, and characterize a protein of interest. This was done over a three lab session experiment that consisted of recombinant DNA used to express a fluorescent protein which was later isolated and analyzed using nanodrop spectrophotometric data. It was conclude that we had expressed the correct protein of relative purity. In future VDS research these skills will become helpful when in need of expressing a protein of interest in the lab and introducing that protein to the top predicted ligands generated by protein-ligand docking software. This will provide vital data on how well a drug binds to a protein based off of software simulations.

__ References: __ [1] Haghi F., Peerayeh S. N., Saidat S.D., Cloning, expression and purification of outer membrane protein PorA of Neisseria meningtidis serogroup B, //Journal of Infection in Developing Countries,//**2011,** 5(12), p856-862

<span style="font-family: 'Arial','sans-serif'; font-size: 13.3333px;"> [2] Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards, A.; Vincentelli, R.; Cambillau, C., Protein production and purification. //<span style="font-family: 'Arial','sans-serif'; font-size: 13.3333px;">Nature Methods //**<span style="font-family: 'Arial','sans-serif'; font-size: 13.3333px;">2008, **//<span style="font-family: 'Arial','sans-serif'; font-size: 13.3333px;">5 //<span style="font-family: 'Arial','sans-serif'; font-size: 13.3333px;">, 135-46.