KHADY+D

__ Title: __ //**You Down With EPC? Yeah for This Protein!**//

//**Well done Khady!**//

__ Introduction: __

Each individual protein has its own set of strategies and protocols that must be worked out in order for purification to occur. [1] Various factors that can affect the purification process include lysis method and conditions, buffer composition, temperature, and protein solubility. [2] During the actual process, the cDNA has to be obtained and the protein has to be expressed of the strain in which you're testing (ex: BL21 (DE3)). [1] The expression happens at a low temperature between 15-25*C in rich medium. [1] Then the protein has to be solubilized and purified in a well buffered solution. [1] IMAC is then used as the initial purification step, then the affinity tag may be removed to achieve further purification and a recombinant hexahistidine-tagged protease is used to reapply the sample to the IMAC column to remove the protease and any cellular proteins that bind to the metal affinity resin. [1] The objective of this lab is to grow a purple protein, purify it, then through characterization, end up with the same protein that we started off with. If all of the instructions are properly followed and the protein itself is left after characterization, then we should see the same molecular weight in the gel using the PAGE ladder, as the one that we looked up.

__ Materials & Methods: __

To express the protein, the E.coli BL21 DE3 was transformed with the plasmid pGEM-gbr22. The bacteria was added to transformation tubes, then underwent a heat shock process. SOC media was then added to it to prevent contamination, incubated, then the plates that the bacteria were on were stored over night in an incubator. The next day, ampicillin and LB were added to a tube, then a single colony of bacteria was picked from our LB/agar plate and transferred into the media. The tube was then placed in a shaking incubator for 8 hours. Later on that day, fresh LB was transfered to a flask and some of the starter culture was transferred from the tube to the flask, then the flask was incubated. The next day, the protein was centrifuged using the Allegra X-15 Centrifuge in order to get the purple pellet at the bottom of the tube. 1 x PBS and lysozyme were then added to the pellet and stored in the -20*C freezer. To purify the protein, we added Cyanase to the tube containing the lysate, then centrifuged it. A syringe filter was then used to remove particles from the lysate. Then 0.5 mL of the Ni-NTA buffer was added to the protein. We then used a Bio Rad chromatography column to and the buffer containing 250mM imidazole to get the protein in Elution 1. We then used a Nanodrop spectrophotometer to determine the concentration of the protein. To characterize the protein, we used an electrophoresis module. We set it up but pouring a 1x TGS buffer into the tank, then we loaded our gels, and used a needle to insert samples 1-6, then samples 4-6 (partner's) into the gel. The gel was then stained, dried, and used for further analysis.

__ Results: __













//Beer's Law Calculations://

For 280nm; .057/118,300(1) = 4.8 x 10-7 mol/L x 25, 974.2 g = .01238 g/L x 1L/1000mL = 1.2358 x 10-5 g/mL x 1000mg/1g = **.01238mg/mL > concentration at 280 nm**

For 574 nm: .086/118,300(1) = 7.3 x 10-7 mol/L x 25,974g = .01896 g/L x 1L/1000ml = 1.896 x 10-5 g/ml x 1000mg/1g = **.01896 mg/mL > concentration at 574 nm**

__ Discussion: __

Based on the PAGE ladder ruler, the molecular weight of my protein is about 30kDa. This is close to the official weight of the protein that we looked up, which was 25,974 Daltons. There was only one protein band present in the Sample 5 lane, so I estimate that the purity of my final protein was 95%. We use lysozyme to digest the cell wall of the E.coli. We used cyanase to degrade nucleic acids. They're both used to separate the protein from the cell. We use the HIS tag system to get the protein in its purest form. After the protein is lysed, it still contains many other proteins from the bacterial host. The Ni-NTA buffer mix is added because the histag of the protein binds to the nickel. Then the PBS buffer is used to remove the proteins that didn't interact with the nickel. Once the protein is bounded, it's eluded using the 250mM imidazole buffer and located in Elution 1. Sample 1 contained our culture before we saw the pellet by centrifuging and before we added anything to our culture. Sample 2 contained the soluble proteins. Sample 3 contained the protein's that didn't bind to the resin. Sample 4 contained the step where we washed the NTA resin with 5ml of 20mM imidazole. Samples 5 and 6 just contain our protein, which is why they were used to base our calculations. The wash buffer contained the 20mM imidazole buffer that we made, but the elution buffer contained a stronger concentration of imidazole buffer, which was 250 mM. There were many possible sources of error throughout the lab. During expression, we were told to sterilize everything and keep the burner on when handling the bacteria, but sometimes, we'd forget to sterilize the pipette tip by running it over the flame. Also, when we were told to gently swipe 1 starter colony into our tubes, I may have swiped more than one colony. During purification, we might not have exactly pipetted right when we made our buffer. The pellets might have been disturbed when we transferred the liquid supernate from the tubes into the 15ml conical tube. The nanodrop that we were using may not have completely been clean, so there was also a risk of contamination there. Lastly, during characterization, we accidentally poked a hole in our gel while pipetting. This led to lane 8 being ineffective and we had to leave it empty. Also, we accidentally tore the side of our gel when we were trying to rinse off some of the dye.

__ Conclusions: __

In this 3 part protein lab, we expressed, purified, and characterized our purple protein. We added DNA, bacteria, ampicillin, and LB media to an organism, which led to us growing a purple wet pellet which we call our purple protein. To purify the protein, we added lysate to the original solution then filtered it using wash and elution buffers. This got rid of everything that wasn't the protein including E. Coli and cell debris. To characterize the protein, we used the gel electrophoresis module to predict the weight of our protein. My gel only had one band in the lane that held sample 5, so my results were pretty accurate. The protein that was characterized in the end was the one that we wanted and the molecular weight of my protein was found to be close to the molecular weight of the actual purple protein. In the future, we will use the skills that we learned in this lab to test future proteins. The characterization basically confirms that you're testing the right protein by using the ladder to make sure that the molecular weights line up. The purpose of this lab is confirmation, which will be very important in our future labs because you always want to make sure that you're testing the right protein.

__ References: __ 1. Methods N. Protein production and purification. //Nature Methods.// **2008.** //2,// 135-46. 2. European Molecular Biology Laboratory. Protein Expression and Purification Core Facility. http://www.embl.de/pepcore/pepcore_services/protein_purification/index.html (accessed April 17th, 2013)