Stephanie+B

__ Bacterial Protein E.P.C (Expression, Purification and Characterization) __
__ Introduction: __

Expression, purification, and characterization steps are used to obtain a sufficient amount of proteins in a medium that can be analyzed. The first step, expression, involves cloning of the target gene to be used to overexpress the protein of interest in a bacterial host [1]. This expression technique can be used to study human proteins without needing human tissues. The pGEM-gbr22 plasmid that is used encodes for a fluorescent protein that makes the solution purple and can be detected by using a UV spectrophotometer to measure light absorbance. The second step, purification, involves separating the protein of interest (gbr22) from the other cellular proteins. The protein is then released from the Ni-NTA agarose by adding imidazole. To maximize the purity of the protein sample, the column should be loaded using a slight excess over the predicted binding capacity [2]. The final step is to characterize the protein. This can be done by using a gel electrophoresis technique in which the protein samples are introduce at the top of a well and move towards the positive end of the electrical supply. The objective of this series of three labs was to analyze a protein obtained through expression and purification techniques. The concentration of the protein, found using the purity and yield of the final product, will correlate with the data found in the spectroscopy measurements. __ Materials & Methods: __

Lab coats, goggles, and gloves were worn as safety precautions while working with the bacteria. Aseptic techniques were also used as safety precautions. While working with the bacteria, a gas burner was turned on. 25 microliters of //Escherichia coli//(DE3) were added to each of 2 transformation tubes. 1.29 microliters of the pGEM-gbr22 plasmid were pipetted into the DNA tube. The tubes were then placed on ice for 30 minutes, heat shocked in 42 degree Celsius water for 45 seconds, then placed on ice for 2 minutes. 200 microliters of SOC media was added. The tubes were then shaken in an incubator for 30 minutes at 250 rpm. 6 colirollers were used to plate the bacteria; 50 microliters of the DNA transformation tube solution was put on the DNA plate while 50 microliters of the control solution was plated on the other plate. A third plate with bacteria from the men’s restroom was used for “fun”. The plates were stored inverted in a 37 degree Celsius incubator for one night, then transferred to a 4 degree Celsius fridge for the second night. In the morning, 0.01 milliliters of 50 mg/ml ampicillin stock was added to two 14 milliliter round bottom conical tubes with 5 milliliters of LB in each. A sterile pipette tip was used to pick a single colony of bacteria and placed in the LB. The tube was placed in the shaking incubator and grown for 8 hours at 37 degrees Celsius and 250 rpm. 25 milliliters of LB were added to a 125 milliliter Erlenmeyer flask and 0.05 milliliters of ampicillin was added. 0.625 milliliters from the starter culture was added to the flask which was again put in a 37 degree Celsius shaking incubator. 500 microliters were put in an Eppendorf tube as sample 1. The Allegra X-15 (Beckman Coulter, Inc, Brea, CA) centrifuge was used to spin a 50 milliliter conical tube filled with the solution for 10 minutes at 5,000 rpm. The liquid was then decanted. 2.5 millilters of 1x PBS and 50 microliters of stock lysozyme were added to the conical tube and it was vortexed and stored in -20 degree Celsius freezer. After a week, the solution was incubated for 20 minutes at room temperature. 2 microliters of Benzonase (Sigma-Aldrich, St. Louis, MO) was added to the conical tube. The lysate was distributed to several 1.7 milliliter microcentrifuge tubes and centrifuged for 20 minutes at 14, 000 rpm at 4 degrees Celsius. 50 microliters of the supernatant was used was sample 2 and placed in the fridge. The rest of the supernatant was then pipetted into a 15 milliliter conical tube. A Wash buffer and Elution buffer were made and kept on ice. The supernatant was then syringe filtered through a 0.22 micrometer PES syringe filter. 0.5 milliliters of Ni-NTA was added and incubated for 15 minutes while a 20 milliliter Econo column (Bio-Rad, Hercules, CA) was set up. The resin and buffer were added to the top of the column an allowed to flow. 50 microliters of this was used as sample 3. The wash step was used as sample 4. The Ni-NTA resin was eluted with 5 milliliters of the buffer with 250 millimolar of imidazole which was collected in a 15 milliliter conical tube as Elution 1. This was done again and labeled as Elution 2. 50 microliters of each Elution 1 and 2 were used as samples 5 and 6. The column was then stripped to be reused. A spectrophotometer was used to measure the absorbance of Elution 1 at 280 nanometers an at 574 nanometers. After another week, sample 1 was microcentrifuged for 5 minutes at 5,000 rpm, the liquid was removed and 200 microliters of nanopure and 40 microliters of loading buffer were added. For samples 2-6 10 microliters of the loading buffer were added. All tubes were deated at 95 degrees Celsius in a heat block for 5 minutes, then centrifuged for 2 minutes at 5,000 rpm. The gel electrophoresis was set up and the tank was filled with 1x TGS buffer. Each lane was cleared using a 20 gauge needle and syringe. 7 microliters of a prestained protein ladder ( Fermentes, SM0671) were added to the first lane. Lanes 2-7 contained samples 1-6 and lanes 8-10 contained a partner’s samples 4-6. It was run for 25 minutes at 200 volts. The gel was stained and after 24 hours was placed on Whatman filter paper (GE healthcare, Maidstone, UK), covered with saran wrap and was dried.

__ Results: __



Protein yield:
For 280nm: A=εbc 0.895=38850 m-1cm-1 (1cm)(c) c=2.304 x 10-5 mg/ml 5ml(2.304 x 10-5 mg/ml) = 1.152 x 10-4 mg

For 574nm: A=εbc 0.895= 11830 m-1cm-1 (1cm) (c) c= 7.566 x 10-5 5ml (7.566 x 10-5 mg/ml) = 3.783 x 10-4 mg



__ Discussion: __

The MW standards were used as a reference to show that the purified protein was about 25 kDa. Lysozyme is used to lyse the cells by digesting the cell walls of the bacteria. Benzonase is used to digest DNA and RNA. Thus, lysozyme and benzonase are used to extract the protein from the bacterial cell. Sample 1 contained the bacterial starter culture in the LB. Sample 2 contained the supernatant after benzonase was added. Sample 3 contained flow waste from the Econo column. Sample 4 contained the protein after the wash solution had been run through. Sample 5 contained the first elution of the protein and sample 6 contained the second elution. The elution buffer was different from the wash buffer because it contained a high concentration of imidazole which stripped the protein. The HIS tag system involves using a histidine affinity tag to tag a protein so it can be tracked while being cloned, expressed, and purified. Possible errors made in these labs include miscalculated masses and solutions made of incorrect concentrations. The first time the elution was run, the elution buffer did not include any imidazole so a new elution buffer had to be made. __ Conclusions: __

These labs were designed to overexpress gbr22 in bacterial cells, and then use them to purify the protein so it could be analyzed using spectroscopy and gel electrophoresis. The concentration of the protein at the maximal wavelength was found to be 2.304 x 10-4 mg/ml. These same techniques can be used in future labs to purify different proteins. Next steps in research include further purification of the protein and crystallography of the protein for ligand binding analysis using virtual screening techniques. __ References: __ [1] European Molecular Biology Laboratory. Protein Expression and Purification Core Facility. [] (accessed 4/16/2012). [2] Nat Methods. **2008 ** Feb;5(2):135-46. Protein production and purification.