Divya+G

=Expression, Purification, and Characterization of the pGEM-gbr22 Protein Using //E.coli//=

__Introduction:__

Recombinant protein is often used to study various biological processes at the enzymatic level[2]. This process includes the processes of finding prospective ligands to these enzymes. It is important to produce a lot of protein to run these tests. //E. coli,// the bacteria used to produce the protein, can be forced to produce the pGEM-gbr22 protein through manipulating lacO promoter to permanently be on [1, 2]. Then, lysate is added to break down the cell to harvest the protein. The protein can be harvested and purified through the use of Ni-NTA which sticks to the histadine tail on the protein and pulls the protein out of the solution. Finally, a gel electrophoresis is run to characterize the size and purity of the protein. If only one band appears, then the protein is a pure substance.

__ Materials & Methods: __

The first step of this experiment is Expression. An agar plate of //E. coli//BL21(DE3) was grown, by adding 25ul of bacteria and plasmid. Then a colony was inoculated into LB-Ampicillin media and placed in a water shaker at 37 degrees Celsius at 200-350 rpm. After a few days, if the protein was expressed, the LB-Broth will turn purple which is the color of the protein. A 500ul sample was taken and labeled sample 1. Then, 50mL of the contents of the cells were distributed into two conical tubes and centrifuged using an Allegra X-15 benchtop centrifuge (Beckman Coulter, Inc., Brea, CA). The fluid was removed from the tube and the pellet was saved. 2.5 mL of 1x PBS solution and Lysozyme were added to the tube which was stored in the -20 degrees Celsius freezer.

The second step if this process of this process is to harvest and purify the protein that was grown in the first step. In order to harvest and purify the protein, the cells must be broken down and discarded. 2ul of benzonase, was added and the lysate from the first step was centrifuged once more. This time, the pellet at the bottom contained cellular waste and was discarded. The remaining liquid is rich in protein and was filtered through a PES syringe filter. This was then added to an Econo column and Ni-NTA was added. In the first wash, elution buffer was added to remove any excess Ni-NTA which did not stick to the His- tag in the protein. The second elution contains pGEM-gbr22, since it removes the protein. To clean the column, 1ml of ethanol(30%) in nanopure water was added. The absorbance of the protein from elution 1 was measured using a Nanodrop spectrophotometer.

The third and final step is to characterize the protein. Through out the lab, various samples were taken at different stages in the protein purification step. THrough the use of gel electrophoresis these samples were analyzed for protein purity. 6x gel-loading buffer was added to each of the 6 samples, and the samples were heat blocked for 5 min at 95 degrees Celcius. SDS page gel was assembled and prepared so that each of the wells held one of the samples and the first well had the DNA ladder from Page Ruler Pre-stained protein ladder. The gels were stained with Imperial dye after it was removed from the assembly, and washed. The gel was placed in the orbital shaker for 90 min. The gel was rinsed and a Kimwipe was added to soak excess dye. and was left in the shaker overnight. The gel was then dried after it was placed on Whatman paper and covered in saran wrap.

__ Results: __

__Fig.1.__ Fun plate from protein expression. The magnetic stir bar remover was swabbed.

__Fig. 2.__ The control plate with no ampicillin is shown above. No bacteria was grown, however there is a lot of condensation.

Fig. 4 The large culture of //E.coli// BL21 (DE3) with the protein pGEM-gbr22 expressed is pictured above. The culture contains LB media and 100g/mL of ampicillin.



Fig.5. The tube was centrifuged and the pellet is shown above

Fig. 6. The spectrophotometry results for Elution 1 (sample 5) from the Nanodrop at wavelength 280. The absorbance was found to be 0.07mg/ml.

Fig. 7. The spectrophotometry results for Elution 1 using UV/VIS Mode in the Nanodrop. The readings for absorbance were taken at 280 nano-meters and 574 nano-meters.

__ Calculations: __ A=Ebc c=A/(E*b)

Concentration at 280 nm:

c=.07/(38850 (M*cm)^-1)(1 cm) = 1.801E-6 mol/L

concentration at 574 nm: c= .046/((118300 (M*cm)^-1)(1 cm)) = 3.888E-7 mol/L

Fig. 8. Results of gel electrophoresis are shown after drying the gel for about 1.5 hours. The wash sample and the elution 1 sample may have been switched since elution 1 would be more "pure," which means that there would be less bands on the elution.

__ Figure 9: __ The Page Ruler ladder is shown above with the potential measurements of the protein in daltons. According to the ruler, the protein is approximately 26 daltons.

__ D ____ iscussion: __ There are many possible sources of error in this lab. First of all, the bacteria used from the company Sigma, was from three years back, so whether or not the bacteria were even alive was debatable. In fact, the bacteria had to be re- inoculated, since the plasmid did not transform the first time. The amount of protein produced was very low as a result of this. The absorbance spectrum from the nano-drop spectrophotometer shows that the the protein had absorbance of 0.060 at 280nm, which, after calculations shows that the concentration of protein is 1.801E-6 mol/L, a very low concentration. Despite these setbacks, the protein was quite pure. The results of the gel electrophoresis show that sample 6, the protein sample had only 1 band on the gel, which is only the pure protein. There might be a slight error when loading the samples since the wash sample, looks more pure than the first elution sample. The wash would contain more impurities and less protein than the wash buffer.

__ Conclusions: __ The protein pGEM-gbr22, was over expressed by the bacteria //E. coli,// through the use of a plasmid that was taken up by the bacteria. This protein was purified through running through a column, and then characterized through the use of gel electrophoresis. All of these steps are important to test potential ligands for a protein. In order to test for a potential ligand, the protein must be pure, which means that the gel must only have 1 line. However, this is not an absolute test of purity, since proteins can be dimers, which can show up as 2 lines on the gel electrophoresis. In this case, NMR or some other form of testing for purity must be used.

__ References: __ [1] Graslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; Protein production and purification. // Nature Methods //. ** 2008 **, 5(2): 135-46. __[2]__ Weill Cornell Medical College. Studying Proteins and Protein Purification. [] (accessed Apr 16, 2012)