Suman+A

__Introduction:__
Due to science and modern technology today, proteins that were once unobtainable can be easily grown and experimented on in a laboratory. The process, called E.P.C. has three distinct parts: expression, purification, and finally, characterization [1].First, the protein is taken from a cell in which it is already, and expressed. For this case, lab grown strain of E. coli bacteria was used to produce protein for experimental work. The bacteria cultures are then allowed to grow and incubate for a necessary amount of time. Next, once there exis t cells with the necessary protein in the lab, they are lysed. The remains of the experiment are carefully filtered out so that only the protein remains at the bottom of the filtration system and all the other parts of a cell are released. During characterization, the protein can be analyzed further using nanodrop spectrophotometry and gel electrophoresis, so conclusions can be made about how it generally works. In this lab, the gbr22 protein was used for protein expression, purification and characterization. Originally isolated from the Great Barrier Reef, the purple gbr22 was isolated and grown, purified, and then characterized [2]. In the end, the data collected throughout the 3 steps was used to determine how pure the protein was and how characterizing other similar proteins could be beneficial in virtual drug screening.

__ Materials & Methods: __

During the lab, it was important to use equipment wisely, and wear a lab coat, goggles, and gloves at all times. The first part involved transforming competent cells, which was down by growing the E.coli cultures to express the purple protein, harvest the cells by centrifugation, resuspend them in buffer, and store them at -20C.For this lab, Sigma bacterial cells were used from the -80 C freezer and three plates, experimental, control, and fun, were created. 200ul of SOC mdia was added and it was shaken in the incubator for 30 minutes at 37C at 25- rpm, and allowed to roll around the plate with colirollers so the bacteria could spread. Each plate had 50 ul of bacteria/SOC mixture. Overnight, the bacteria on the plates grew at 37C. 10ul of amipicillin were added to two 5ml tubes of LB in a sterile culture tube with a cap. They were taken from a starting culture grown with 100 ug/ml ampicillin. A single colony of bacteria was picked and gently dipped into the LB. These were grown for 8 hours at 3C at 200-350rpm. The plates were then stores in 4C fridges.The media in the flask turned a purple color after shaking in the incubator for 16-24 hours at 100ug/ml concentration of both ampicillin and LB broth. 0.625 ml of the starting culutre was transferred into 125 ml flasks and the cells were ready to be harvested. After centrifuging for 10 minutes at 5000rpm and 4C, the cell pellets were at the bottom of the tubes.During protein purification a filtration system was used to get the overexpressed purple protein away from the rest of the material in the cell. The E.coli cells were broke open, and .5ml Ni-NTA and 5ml of 20mM imidazole was used to purify the protein from all the other molecules attached to it. In the immediate step, the concentration of imidazole increased to 250mM. Over the course of the purification, six small 50ul samples were taken in each step to be analyzed later in gel electrophoresis, and the final protein was stored in the freezer.Next, the nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) was used to measure the maximal wavelength of the protein at a standard wavelength and its maximum, according to data from past research. Gel electrophoresis was then performed on the protein to show that it had, in fact, became pure.

__ Results: __ Fig 1: Two cultural plates, both with ampicillin, were grown. The purple plate also included bacterial organism BL21 (DE3) and pGEM-gbr22 and shows that the protein was expressed successfully. The second plate looks clear because the ampicillin did not allow any growth to occur, and therefore the plate served as a control. Fig 2: 0.625 ml of the starter culture were placed in an Erlenmeyer flask with LB media and Ampicillin (final concentraltion of 100 ug/ml) and allowed to grow for 16-24 hours at 37 degrees Celsius and 200-350 rpm Fig 3: After the bacteria had been allowed to grow, the solution was centrifuged at 10 minutes at 5,000 rpm at 4 degrees Celsius and the purple protein sank to the bottom. It weight 0.19 grams. Fig 4: Elution 1 was retained in a 15 ml conical tube after the grb22 protein was released by using Ni-NTA resin, which had a high concentration of imidazole. Fig 5: After the imidazole was allowed to release the protein from the rest of the molecules in the cell, Elution 2 was taken to be sure that the protein really had been purified. Fig 6: Trial 1 for the gbr-22 protein from Elution 1 in the Nanodrop showed an absorption of 0.753 at a wavelength of 280 nm. n=2 Fig 7: Trial 2 for the gbr-22 protein from Elution 1 in the Nanodrop showed an absorption of 0.724 at 280 nm.n=2 Fig 8: Trial 1 for the US-Vis setting in the Nanodrop measured the Elution 1 protein at a standard wavelength of 280 nm and at its maximum wavelength of 574 nm. The absorption rates were 0.061 and 0.112, respectively.

Fig 9: Trial 2 for the US-Vis setting in the Nanodrop measured the Elution 1 protein at a standard wavelength of 280 nm and at its maximum wavelength of 574 nm. The absorption rates were 0.073 and 0.089, respectively. Fig 10: Gel electrophoresis was performed on each of the samples taken during the purification of the gbr-22 protein and it confirmed that the protein had been purified by the end of the lab. Fig 11: A standard ladder displays molecular weights of known proteins after electrophoresis and can be compared to results in the lab to characterize a specific protein. Beer's Law Calculations:

A= ebc where A is the average absorbance, e is the extinction coefficient, b is the path length, and c is the concentration of the sample.

c= a/(eb)

Using the above formula, concentration of the protein at 280 nm: c= .735/((38850)(1)) c= 1.891e-5

Concentration of the protein at maximum wavelength of 574 nm: c= .845/((118300)(1)) c= 7.14e-6

__ Discussion: __

The results from the lab show that gbr-22 can be overexpressed using E.coli, and then purified out with solutions such as Ni-NTA. We could then confirm that everything but gbr-22 had been filtered out using electrophoresis to find its molecular weight and compare it to known data, which in this case matched. The purple colored protein first grew in the colonies, giving them a purple color, which was enough to see that it was being expressed in the bacterial colonies. It was then incubated and grew some more, giving the solution a purple color as well. The more purple the color, the more the protein concentration in the solution.

During purification, gbr-22 was separated from everything else in the cells and 6 samples were taken along each step of the purification process. By using lysozyme and Benzonase, most of the cell material was released, but the stuff bound to the gbr-22 was not coming off. Therefore, Ni-NTA was used to completely get rid of everything but gbr-22. This can be seen in the electrophoresis results above, where some contamination exists along the 5th bar, but as soon as Ni-NTA is added, the protein is completely free.

In the gel results above, Sample 1 contained the protein with everything else in the lysed cell, soluble and insoluble. In sample 2, lysozyme and Benzonase had been added, so all the soluble matter had been filtered down. In Sample 3, nothing new was added, but the filter was allowed to run just so more loose substances could come out. In sample 4, 5ml of 20mM imidazole was added, which released most all of the extra proteins tied to the resin. For solution 5, an even higher concentration of imidazole was added so that the protein could be completely free.

The wash buffer is different from the Elution 1 because a smaller concentration was used in the wash when the filtration occured. Elution 1 had a higher concentration of imidazole because it was needed to release gbr-22, which has 6 HIS (histidine) tags. During the wash stage, the proteins with less HIS tags were filtered out, but the gbr-22 was attached to the nickel beads. Adding a stronger concentration of Imidazole, which also attaches to the beads, provided competitive binding, in which the Imidazole "outcompeted" the gbr-22, therefore releasing it from the beads.

Because the labs were spread over 3 weeks and the work was detailed, there were many possible errors made, the most significant being sanitation when working with the delicate proteins. It wasn't easy to always work rapidly and still be consistent and careful with the techniques listed in the protocol, so the tubes and flasks were sometimes left open longer than they would have been ideally. Air could have contaminated many of the procedures as well, especially during picking colonies or transferring solutions from one container to another.

__ Conclusions: __

In this lab, the overall purpose was to overexpress a recombinant protein in E. coli, to purify the gbr-22 protein from the rest of the cell matter, and then analyze the protein and its different purification stages through spectrophotometry and gel electrophoresis. The results of the electrophoresis showed that the protein had been purified successfully. In the future, protein expression, purification, and characterization can all be used in virtual screening when researchers work with a new protein and want to isolate it from the rest of the material to perform experiments on it in the lab.

__ References: __