Andrew+B

__ Title: __ Understanding the Nature of Proteins Through the Use of Expression, Purification, and Characterization Methods

__Introduction:__ A popular method of obtaining protein for study is to transform a host cell, in this case a bacterial cell, with a plasmid modified with the appropriate sequence for expression of the protein in the appropriate host cell [1]. In order to have the bacteria take up the plasmid and incorporate it into the genome, two methods are predominantly used: electroporation and heat shock [2]. //E. Coli// is a common host for recombinant protein production because it is inexpensive, throughly studied, and optimized for a variety of functions[1]. The BL21(DE3) strain is particularly useful for the purpose of protein production because it lacks the lon and ompT proteases and is complementary with a common promoter system [1]. This experiment will utilize the heat shock method. To facilitate the screening of proteins, the sequence in the transformation vector is often altered so that the protein would be expressed with an extra six histidines on the N-terminus that will allow it to be captured using chromatography techniques. Once the protein has been extracted and purified, it is often characterized using gel electrophoresis and spectroscopy to determine concentration and molecular weight of the protein. In this experiment, the coral protein gbr22 will be expressed in bacterial cells, purified for study, and then characterized. It is hypothesized that the gel that will be obtained will show the molecular weight of the gbr22 protein to be around 25,000 kD, close to the accepted value.

__ Materials & Methods: __ Three plates were created: a experimental plate containing transformed bacteria, a control plate, and a "fun" plate that was coughed on. To create the experimental plate, the pGEM-gbr22 plasmid was used to transform //E. Coli// BL21(DE3) (New England Biolabs, Ipswich, MA) using a heat shock method. These were grown in SOC media on agar plates and incubated at 37C overnight. Pictures were taken the next day (Figures 1a-c). A starter culture was grown in LB with ampicillin and this was used to grow a large culture. Sample 1 was taken from a purple large culture flask (Figure 2). The culture was spun down using an Allegra X-15 Centrifuge (Beckman Coulter, Inc, Brea, CA) and the pellet was resuspended in a 1X PBS solution to which lysozyme was added (Figure 3). Benzonase was added and the lysate was clarified using small centrifuges. Sample 2 was taken from the supernatant. The soluble fraction was isolated and syringe filtered. The protein was then purified using batch and column chromatography with a Ni-NTA resign/buffer. A sample was taken from the flow through of the chromatography column (sample 3), one from the wash step which used 20mM imidazole (sample 4), and one from each of two elution steps done with 250mM imidazole (samples 5, 6) (Figure 4). A Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) was used to determine the concentration of protein in sample 5 (Figures 5-8). Electrophoresis using a Mini-PROTEAN tank with a standard ladder (Figure 10) and samples1-6 was run. The gel was then removed, stained, and dried (Figure 9). It was analyzed for purity and molecular weight.

__ Results: __

Figure 1a: Transformation Plate Containing Around 650 Colonies of //E.// //Coli// BL21(DE3) Transformed with pGEM-gbr22 Plasmid Figure 1b: Control Plate Containing No Bacterial Colonies Figure 1c: Fun Plate From Cough Sample with a Single Colony

Figure 2: Large Culture of //E.// //Coli// BL21(DE3) Transformed with pGEM-gbr22 Plasmid in 125ml Erlenmeyer Flask Grown in LB and 100g/ml Ampicillin

Figure 3: Cell Culture Pellet of 0.59g Resuspended in 1X PBS Solution

Figure 4: Elution 1 and 2 After Batch and Column Chromatography with a Ni-NTA resign/buffer Eluted with 250mM Imidazole Figure 5: Trial 1 Spectrophotometry Results from Nanodrop in Protein A280 of Elution 1 (sample 5) at a Wavelength of 280nm. Absorbance Value of 0.75.

Figure 6: Trial 2 Spectrophotometry Results from Nanodrop in Protein A280 of Elution 1 (sample 5) at a Wavelength of 280nm. Absorbance Value of 0.80

Figure 7: Trial 1 Spectrophotometry Results from Nanodrop in UV/VIS Mode of Elution 1 (sample 5) at Wavelengths 574nm and 280nm. Absorbance Values of 1.13 and 0.83 Respectively. Figure 8: Trial 2 Spectrophotometry Results from Nanodrop in UV/VIS Mode of Elution 1 (sample 5) at Wavelengths 574nm and 280nm Absorbance Values of 1.30 and 0.81 Respectively.

__Beer's Law Calculations for Concentrations in Elution 1 (Sample 5):__ Beer's Law: A=Ebc

Yield at 280nm Wavelength A=0.80, a=38850 M-1 cm-1, b=1cm c=A/Eb=2.059x10-5 M (25794.2g/mol)= 0.53 mg/mL Yield=cV=(0.53mg/ml)(4.4mL)= 2.34mg

Yield at 574nm Wavelength A=.1215, a=118300 M-1 cm-1, b=1mm c=A/Eb=1.03x10-5 M (25794.2g/mol)=0.27mg/ml Yield=cV=(0.27mg/ml)(4.4mL)=1.19mg

Figure 9: SDS-PAGE Gel Electrophoresis Result. Lanes from Left to Right: Molecular Weight Standard (Fermentas 00070544 #SM0671), Samples 1-6 for First Researcher, Samples 4-6 for Second Researcher (Ignore Due to Well Contamination).

Figure 10: Fermentas PageRuler Pre-Stained Molecular Weight Standard (00070544 #SM0671). Measurements in KiloDaltons.

__ Discussion: __ It was found through gel electrophoresis techniques that the molecular weight of the protein was between the 26 and 34 kD marks on the molecular weight ladder, estimated at around 28 kD. The purity was estimated to be around 75% based on the number of other bands and the relatively low intensity of these bands compared to the band of the protein in question. Lysozyme was used in this lab to break down the cell walls of the bacteria, lysing the cells and mixing the contents. Benzonase was then used to reduce the viscosity through the digestion of DNA and RNA in the mixture. Sample 1 contained bacterial cells harvested directly from the large culture flask. Sample 2 contained the supernatant remaining after lysing and clarifying, containing soluble proteins. Sample 3 was taken from the first flow through of the chromatography column and accordingly contained all proteins that didn't interact and bind to the HIS tag. Sample 4 was taken from the wash with 20mM imidazole and contains only loosely bound proteins, not gbr22. Sample 5 and 6 were taken from the elusions with 250mM imidazole and contain the gbr22 protein along with a few contaminant proteins. The difference between the wash and elution buffer is the concentration of imidazole used. The wash used 20mM imidazole to remove loosely bound proteins while the elution used 250mM to remove all proteins from the column. The HIS tag system works through the use of a Ni-NTA resign that captures and holds proteins with histidine tails which bind the nickel ions. This allows the gbr22 protein, which has been modified to have an extra six histidines on the N-terminus, to be isolated from the rest of the cellular proteins.

__ Conclusions: __ In conclusion, a modified gbr22 protein sequence was cloned into bacterial cells, over-expressed, purified, and characterized. It was found that the molecular weight of this protein is around 28kD with a purity in our sample of about 75%. Some sources of error included contamination of the last three wells in the gel and variation in the concentration chosen for analysis from Elution 1 that could lead to different results in the analysis of absorbances. In the future, the methods and techniques employed in this series of experiments will be used to study a novel protein that will be targeted for drug discovery. These methods can be used to obtain the appropriate amount and concentration of protein needed for crystallography studies of proteins that may be necessary to target certain proteins essential in various infectious diseases.

__ 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] Protein Expression and Purification Core Facility. [] (accessed April 16).