ALYSSA+K

= Production of gbr22 Protein =

__ Introduction: __ There are many choices to be made when determining a protocol for producing a purifying a protein. No one protein is the same, so there are many techniques in which this can be done [1]. Most often, individual attempts at producing protein end in failures, but the overall technique has been successful [1]. The bacterium used in this lab, E. coli, is expressed and purified with a His6 tag to be used in the purification process [2]. But depending on the protein and bacteria being used, different tags will be used [2]. During this set of experiments, gbr22 protein will be produced and purified so that no other proteins remain. The protein will then be characterized to confirm that the sample is pure. The protein produced will not be contaminated with other non-target proteins.

__ Materials & Methods: __  The first part of this lab consisted of producing the target protein, gbr22. On the first day plasmid DNA was added to competent E. coli cells which were then heat-shocked and incubated in the shaking incubator for 30 min at 37 ° C and 250 rpm before being place onto agar plates and the stored in the incubator at 37 ° C overnight. The next morning, a single colony was picked from the plate and transferred to LB/amp media and placed in the shaking incubator for about 8 hours at 37 ° C and 200-350 rpm. That evening, 0.625 ml of the starter culture was transferred to a sterile 125 ml Erlenmeyer flask with fresh LB media and ampicillin of concentration 100 m g/ml. The flask was incubated for 16-24 hours. The next day, the bacteria was poured into a 50 ml conical tube and centrifuged for 10 minutes at 5,000 rpm at 4 ° C. The supernatant was decanted and the pellet weighed. The pellet was resuspended and lysozyme was added. The second part of this lab was purification. Cyanase was added and then the protein was centrifuged and the supernatant decanted. The protein remaining was then filtered through Ni-NTA resin. 20 mM and 250 mM imidazole was used to remove the protein from the resin. Nanodrop spectroscopy was then used to determine the concentration of the protein. And in the last part of this lab, the protein was characterized by running a gel of the protein from samples during vital steps of this lab.

__ Results: __













A=Ebc Average A= 0.3125 extinction coefficient at 280 nm= 38850 molecular weight of gbr22= 2579402 g/mol 0.3125=38850(1)c c= 8.04E-6 mol/L= 0.207 mg/ml

__ Discussion: __ In this lab lysozyme was used to breakdown the cell walls of the bacteria that was transformed. This would lyse the cells so that the protein from inside could be obtained. Cyanase was used to breakdown the DNA and RNA of the bacteria cells so that when the cells were spun down, only protein would be left in the pellet. The HIS tag is a chain of histidine amino acids at the end of the protein. These amino acids bind with the nickel found in the Ni-NTA resin. So by binding to the resin, these proteins will stay put while other proteins are washed away. When stron concentrations of imidazole are added, the imidazole competes with the histidine to bind with the nickel, and thus the protein is removed from the resin. Sample one is a sample of the bacterial culture and contains all proteins from the bacteria used. Sample two is the supernatant solution obtained after centrifuging the protein after cyanase was added. This sample contains only the protein found in the cell and not the genetic material or other cellular debris. Sample 3 is the waste collected after the first initial flow through of the protein after being mixed with the Ni-NTA resin. This sample contains proteins that did not bind to the resin. Sample 4 was collected after the weak concentration of imidazole was added to the protein-resin mixture. This sample contains any proteins that were loosely bound to the resin. Sample 5 was obtained from Elution one, which contains the proteins that were removed from the resin after being washed with a stronger concentration of imidazole. This sample should theoretically contain only the target protein. And finally sample 6 was obtained from elution 2 which contains any protein left after the first wash with the stronger concentration of imidazole. This sample should theoretically have no protein in it. The wash buffer contained 50 mM of imidazole which was used to only remove proteins weakly bound to the Ni-NTA resin. The elution buffer however contained 250 mM imidazole which removed most if not all proteins bound to the resin. The protein was found to be about 30 kDa or 30,000 g/mol after characterization. The size of the protein used during the purification step was 25,794.2 g/mol. The purity of the sample was estimated to be about 80%. This is due to the light band of a smaller protein that was found in the elution 1 sample. Some sources of error include inaccurate estimations of the protein’s size due to a molecular ladder that could have had more detailed measurements. Another source of error concerning the purity of the sample is not letting the wash step occur slowly enough, so not all proteins were removed and were then remaining in elution 1.

__ Conclusions: __ In this lab gbr22 plasmid was transformed into BL21(DE3) //E. coli// cells which then produced the gbr22 protein. The protein was then purified and characterized. The yield of protein was 1.037 mg and had a purity of about 80% according to the characterization. By knowing these procedures, target proteins will be able to be produced and purified so that only the target protein is being tested. These proteins will then be used to verify the results of the ligand matches from virtual screening for target proteins in wet lab.

__ References: __ [1] Nat Methods. **2008 ** Feb;5(2):135-46 [2] European Molecular Biology Laboratory. Protein Expression and Purification Core Facility. [].

[3] PageRuler Prestained Protein Ladder #26616. [].