DANIEL+D

__ Title: __ Expression, Purification, and Characterization of gbr22 Purple Protein

__ Introduction: __ When possible drug targets and drugs have been determined through virtual screening, the next step is to synthesize a large amount of the target protein for testing. Steps commonly used to initially synthesize and purify these proteins are to first express a target protein in another cell by using recombinant plasmid DNA. This protein must then be purified and removed from the other cellular components. Finally, the resultant protein must be characterized to ensure purity. (great!! - maybe explain what recombinant DNA is?)

Gbr22 was the protein chosen, and the plasmid pGEM-gbr22 was utilized to insert thhttp:vdsstream.wikispaces.com/DANIEL+De recombinant DNA coding for the protein into the chemically competent E. coli BL21 cells. Plasmids are generally loops of DNA that function by entering the host cells and telling the cell to express its protein. (hmm..maybe phrase it differently?) The plasmid used will spur the BL21 to synthesize gbr22. The pGEM-gbr22 plasmid has a gene that encodes ampicillin resistance. In order to ensure that only transformed cells were expressed(grow), the BL21 cells were grown on an agar plate containing ampicillin. This beta lactamase (I think you mean beta lactam antibiotic, not beta lactamase) will kill any non-transformed BL21, cells that do not have the gene coding for ampicillin resistance, while the transformed cells will still successfully grow.

Once the BL21 cells reached log-phase growth, the time when the protein will be optimally expressed, their growth was halted and lysozyme was added. This lysed open the cell membranes and allowed the protein to exit the cell for purification. After the cells were spun down and the supernatant which contained the protein was extracted, it was placed in a column chromatography rig which contained Ni-NTA resin. An important aspect of the gbr22 was that is was tagged with six histidine amino acids which bound to the Nickel beads present in the resin. This allowed the protein to be isolated and purified. Spectrophotonomy (spectroscopy) and Beer's law were then used to calculate the amount of gbr22 purified.

Gel electrophoresis is a process that separates the macromolecules to be analysed. A gel was run to visualize the protein and determine relative purity.(mention PAGE gels)

The objective of the lab was to overexpress the protein Gbr22 in E. coli cells by using the plasmid pGEM-gbr22, lyse the cells open and purify the protein using a combination of batch(?) and column chromatography, then characterize it through gel electrophoresis.

__ Materials & Methods: __ The main sections of the experiment were expression, purification, and characterization.

Expression consisted first of placing 25ul of competent BL21 cells in a transformation tube with 1.5ul of pGEM-gbr22. These were set on ice for 20 min., then heat shocked at 42°C for 45 sec. SOC media was added and the mixture was shaken for 30min. at 27°C. After, colirollers were used to plate the cells on LB with 100ug/ml of ampicillin. The plates were allowed to grow at 37°C overnight. A colony was taken from the transformed plate and starter cultures were grown in LB with 100ug/ml ampicillin at 37°C for 8 hours. Then a large culture was allowed to grow again with 100ug/ml ampicillin at 37°C for 20 hours until the cultures were purple in color. Finally, the bacteria was spun at 5000rpm, the pellet was collected, lysozyme was added, and then it was resuspended in a 1X PBS solution. This was stored in the -20°C freezer until purification.

For purification, the cells were allowed to thaw for 20 minutes to allow the lysozyme to activate. Benzonase was added after to break down nucleic acids and make the solution less viscous. This solution was spun at 14000rpm at 4°C and the supernatant containing the protein was collected. This was placed in a 20ml Bio-Rad chromatography column containing .5ml of Ni-NTA resin. A wash containing 20mM imidazole was run through the column and discarded. 2 Elution buffers were then run through the column containing 250mM imidazole and the flow containing the purified gbr22 was collected. The Nanodrop (Thermo Scientific, Wilmington, DE) spectrophotometer was used to determine the concentration of the protein at 280 and 547nm.

Characterization consisted of running gel electrophoresis with 6 samples taken at periodic intervals during the purification process. The gel was run in 1X TGS buffer using a BIo-Rad precast polyacrilamide gel, and each sample was treated with a 6X loading buffer SDS-PAGE containing bromophenol blue,DTT, glycerol, and SDS. The gel ran for 25 minutes at 200V with a Pagerules pre-stained ladder from Thermoscientific. The gel was then qualitatively evaluated. __ Results: __







Discussion: In Figure 1 the successfully transformed BL21 cells are shown with Figure 2 demonstrating no contamination on the control plate. Elution 1 and 2 show the gbr22 successfully incorporated into the elution solutions. There is some error, however, because Elution 2 is purple indicating that not all of the gbr22 was displaced in the first elution. Elution 2 yielded 1.83mg of gbr22.

During the purification aspect of the lab, lysozyme was used to lyse the cell membranes and allow the protein to exit the cell. Benzonase was also added to break down nucleic acids and make the solution less viscous. The six histidine amino acids added to the gbr22 bound to the Nickel beads in the Ni-NTA resin which allowed the gbr22 to be separated from the other cellular components. To release the target protein from the beads, 250mM imidazole (what exactly does imidazole do?) was added to push the gbr22 off of the Ni in elutions one and two. Samples were taken periodically throughout the lab for use in the characterization gel to ensure the processes were happening correctly. Sample 1 contained all of the cellular components, sample 2 was the soluble fraction after centrifugation, sample 3 was the initial flow through the chromatography column, sample 4 was the wash buffer where 20mM imidazole was washed through the column, and samples 5 and 6 were elutions with 250mM imidazole and the final gbr22 protein. The 20mM wash buffer did not compete with the HIS tag, but the elution buffers did. From the gel it can be seen that the gbr22 was about 27kDa, where its actual weight is 25.8kDa. The sample is about 80% pure because a contaminating band can be seen in well 6 of the gel, however the main band was the gbr22. The contamination indicates error that could have come in at any point of the purification steps. Most likely, a protein was attached to the Ni beads and was not displaced by the wash buffer. As a result it was incorporated in to the two elution buffers.

Conclusions: pGEM-gbr22 was used to express gbr-22 in chemically competent BL21 cells, the cells were lysed then purified as a result of the HIS tag and Ni-NTA resin, and finally the protein was characterized using gel electrophoresis with TGS buffer 1.83mg of the gbr22 was collected from the cells, however it was estimated that it was only 80% pure due to a contaminating protein.

This applies to virtual drug discovery because it is the process used to synthesize target proteins that can be used to test various drugs. Different proteins can be expressed by using plasmids inserted into other cells, HIS tags or other markers can selectively purify the selected protein, and gel electrophoresis can be used to determine the relative purity of the target.

__ References: __ Lorenz, M; Wackernagel, W. Bacterial gene transfer by natural genetic transformation in the environment. Microbiol. Rev.// **1994**. 58(3), 563-602.

ThermoScientific. PageRuler Prestained protein ladder. [] (Accessed April 17, 2013)