JENSEN+G

__Title:__
 * Analyzing Yield and Purity by Overexpressing, Purifying, and Characterizing Gbr22 **

__Introduction__: For practical use in any wet lab that uses proteins, such as VDS for drug screening, an abundant supply of a pure form of the target protein is required. One of the first steps when beginning protein purification is to determine the actual method that will be used to purify it. Over the spectrum of different labs that use proteins, the steps to purify protein can vary greatly. As a good rule of thumb when purifying, as well as most other things in science is to keep the steps as simple as possible, if not it is easier to contaminate your protein you are trying to purify [1]. The protein purification lab that was conducted in VDS was actually a series of three labs: Protein Expression, Protein Purification, and Protein Characterization. The goal of the lab was to overexpress and eventually purify a sample of just the “purple protein” or gbr22. E. Coli cells (BL21DE3) were heat shocked to so they could be injected with the plasmid (pGEM-gbr22) that codes for the purple protein. These cells were then incubated so after a few days a sample of the bacteria with the overexpressed protein could be harvested and centrifuged to form a “cell pellet”. The cells were then lysed and broken apart by adding lysozyme and cyanase to the pellet and centrifuging this. After this step another pellet was obtained, this time with only parts of the cell and the target protein. This was then combined with Ni-NTA and filtered with solutions containing various amounts of imidazole and PBS. Gbr22 binds to histidine tags still in the solution, but when the imidazole is added to it it competes and binds to the histidine tags, detaching the gbr22, causing it to wash out. This solution was then analyzed through spectrophotometry to find out the yield, and then ran through a gel by gel electrophoresis to find the purity of the ending protein solution. By the end of the experiment a sample of purified protein was obtained for further use. Hypotheseis: As you add the imidazole solution, the first wash with a high concentration of imidazole will yield the highest concentration of protein.

__Materials & Methods: __ First the E. Coli cells must be transformed to contain the plasmid that codes for the purple protein. Two transformation tubes were obtained and 25ul of bacteria (BL21DE3) was added to the tubes. Then 2ul of the plasmid was added to one tube, the other was a control, and both tubes were heat shocked in a water bath. After 45 seconds they were placed on ice. 200ul of SOC was added and this was left in the shaking incubator for 30 minutes. Then 50ul of the incubated mixtures were pipetted on AMP LB plates and spread with colirollers. This was stored in the 37 C incubator overnight. The next morning two culture tubes of 10ul ampicillin and 5ml LB agar were mixed. A single colony was picked up from the plate containing DNA with a pipette and the tip was dropped in the amp/agar mixture. These tubes were then placed back in the shaking incubator for another 8 hours. Then 625ul of the culture was mixed with 25ml LB and 50ul ampicillin in a 125 ml flask. This was incubated for another day. Then, a 500ul sample was taken and stored in a eppendorf tube in the 4C fridge (labeled “Sample 1”). The remainder of the flask was poured into a conical tube and centrifuged for 10min. A pellet will form from which the liquid can be decanted from. The cells were resuspended in a 1x PBS solution by vortexing and add 50ul of lysozyme to the mixture. Then 2ul of cyanase was added to the tube and let sit for 15min. This was distributed to several microcentrifuge tubes and centrifuged for 20 minutes. 50ul of the liquid was then taken as “Sample 2” and stored in the fridge. The rest of the liquid was decanted into a tube. A column was set up with the filter and a mixture of .5ml Ni-NTA and the decanted liquid was added. It was filtered into a “Waste tube” (50ul was taken as sample 3). A wash solution (1xPBS+20mM Imidazole) was added and flowed through (Sample 4). Then an elution buffer(PBS+250mM imidazole) was flowed thorough, twice (samples 5 and 6). The elution 1 was analyzed with nanodrop and samples 1-6 were run in a SDS-PAGE gel by electrophoresis. The gel was then stained, destained, then dried.

__Results__:















**__Beer's Law Calculations__** A=Ebc __At 280nm__ Absorbance=.2835=A Extinction Coefficient=38850m^-1c^-1=E Pathlength=1cm=b c=? .2835=38850*1*c **c=7.297e-6** MW=25794.2 g/mol c*MW= gr/liter 25794.2*7.297e-6= **.188 g/l** __At 574____nm__ Absorbance=.555=A Extinction Coefficient=118300m^-1c^-1=E Pathlength=1cm=b c=?

.555=118300*1*c **c=4.69e-6**

MW=25794.2 g/mol

c*MW= gr/liter

25794.2*4.69e-6= **.12 g/l**

__Discussion__: The results from the protein purification lab supported the hypothesis, however the purity that showed up from the electrophoresis run was lower than expected. This could be from many different potential sources of error. Lysozyme was used to break down the cell wall and separate the protein from the rest of the cell, and cyanase was used to break down DNA and RNA so we could further isolate the protein. A common pitfall is having the “pure protein” contaminated with cell debris. This can stem from poor lysis [2]. It is important to take care when separating any pellets from the liquid so the two are not mixed. When the various elutions and wash buffers were ran through the column, the imidazole in the buffer was competing with the protein to bind to the histidine. As the concentration of imidazole was increased, more of it was binding and essentially kicking the protein out into the “flow through”. With this knowledge it is possible to asses what is in each of the samples we took. Sample one contained whole cells. Sample two contained a mixture of various parts of the cell, including the protein but not including heavy cell debris. Samples three and four contained none of the gbr22 protein. Samples five and six (mostly 5) contained the bulk of our purified protein. From the results of the gel, the protein was only purified to about 33%, which is pretty low. Because of the multiple bands, the size of the protein could not be distinguished from the gel.

__Conclusions__: This lab utilized lysing the cell and Ni-NTA purification to make a purified yield of a protein. By analyzing nanodrop and doing a gel electrophoresis run the yield and purity can be found. We can use these volumes in further labs such as protein affinity.

__References__:

[1] Protein Purification Handbook; Amersham Biosciences, pages 7-8

​[2] Graslund, S.; Nordlund, P., Weigelt, J., et. al, Protein production and purification. Nat Methods 2008, 5 (2), 135-46.