BRENDAN+C

__**Title: **__ Purification and Characterization of pGEM-gbr22 protein expressed by recombinant Escherichia. Coli

__**1. Introduction: **__  Proteins of interest are often found only in low concentrations in target organisms, and difficult to isolate [1]. The purification and production of these targets would allow for X-ray crystallography, enzyme inhibition assays, and other techniques to be applied to investigate the attributes of the proteins of interest. The techniques of production and over-expression of proteins have been modified and elaborated upon, but this experiment validated and investigated the expression of a protein found in coral (gbr22) after expressing it in //Escherichia. Coli (E. Coli//) [2]. This method transformed the //E. Coli// and incorporated the pGEM-gbr22 plasmid into its genome. After multiple rounds of division, the //E. Coli// would produce greater amounts of the protein and the next step, purification, would be used to isolate the target protein from the other proteins in the //E. Coli.// Although many different techniques could be used for purification, the pGEM-gbr22 plasmid coded for a chain of 6 Histadines (HIS) to the gbr22 protein. This suggested the use of Ni-NTA Affinity Chromatography in which the 6x HIS tag would tightly bind to the Ni matrix [3]. This technique employs the differential binding strengths of proteins to the Ni in order to wash the more loosely bound, contaminant proteins, out of the purified protein solution. In order to accomplish this, a compound (imidazole) that competes for the Ni sites was used at varying concentrations. Characterization was the final process, and gel electrophoresis was employed to characterize, by mass differentiation, the validity of the purification and expression as applied to the protein of interest. This technique uses charge and voltage to slowly move denatured protein through a gel mesh in order to differentiate between proteins of differing molecular weight (MW).  If each step of the expression, purification and characterization were carefully and accurately performed, the results of the characterization should yield a heavy band that corresponds to the molecular weight of the protein of interest.

__**2. Materials & Methods: **__

//2.1. Protein Expression// 25 uL of //E. Coli// BL21(DE3) (Cite company & state) was transferred to 2 transformation tubes. pGEM-gbr22 plasmid was added to the DNA tube and both tubes were heat shocked at 42 degrees Celsius for 45 seconds. SOC media was added to each of the tubes and were shaken in a water incubator for 30 minutes at 37 degrees Celsius at 250 rpm. 50ul of bacteria/SOC mixture was pipetted from each tube and added to agar plates containing ampicillin. Overnight incubation followed. A single colony of bacteria was then added to LB/ampicillin media. The two tubes were incubated for 8 hours (37 degrees Celsius 200-350rpm). 0.625mL of starter culture containing pGEM-gbr22 was transferred into an Erlenmeyer flask that contained LB and ampicillin. The flask then shook for twenty-four hours in a dry shaking incubator. A 500uL sample of culture was taken (sample 1). The remaining contents of the bacteria were poured into a 50ml conical tube and were centrifuged into an Allegra X-15 benchtop centrifuge (Beckman Coulter, Inc., Brea, CA) and was set to run for 10 minutes at 5000 rpm and 4 degrees Celsius. The purple pellet at the bottom was saved and the remaining liquid was decanted. 2.5 mL of 1x PBS solution was mixed with the pellet. Lysozyme was also added to the tube and the tube was stored in the -20 degrees Celsius freezer.

//2.2. Protein Purification// 2uL of Benzonase was added to the tube that was stored in the -20 degrees Celsius freezer, in order to facilitate DNA and RNA removal. The lysate was distributed into two Eppendorf tubes and were then centrifuged. 50ul of supernatant was then taken (sample 2). The liquid supernatant from both tubes were transferred into a conical tube and filtered though a PES syringe filter (What size). An Econo column was used to run the supernatant along with the Ni-NTA resin/buffer. The supernatant was mixed with 0.5mL of Ni-NTA resin/buffer mix and run through the column (50uL of this was taken as Sample 3). 5mL of Wash elution that consisted of 1x PBS and 20mM imidazole was run through the column (50uL of this was taken as Sample 4). Then, 5mL of elution buffer that consisted of 1x PBS and 250mM imidazole was run through the column twice (first time: 50uL taken as sample 5, and second time 50uL as sample 6). The absorbance of the protein solution from elution 1 was measured using a Nanodrop spectrophotometer (Cite company & state) at 280 and 574 nanometers in order to make sure that the gbr22 protein was purified into the elution solution.

//2.3. Protein Characterization// Characterization was accomplished using gel electrophoresis. From expression and purification, six samples were gathered and placed into a heat block at 95 degrees Celsius for 5 minutes, after which they were centrifuged for 2 minutes at 5,000 rpm. loaded into Bio-Rad precast polyacrylamide gel. T he SDS electrophoresis was assembled and prepared accordingly so that the samples could be run for twenty-five minutes at 200V in a Mini-PROTEAN electrophoresis tank filled with 750mL of 5x TGS buffer. The samples were resuspended in 10uL 6x sample loading buffer and loaded into the gel along with a Page Ruler Prestained Protein Ladder (ThermoScientific). After running for 25 minutes at 200V, the gel was stained using Imperial protein stain and then rinsed with NP water in a shaker overnight. The next day, the gel was dried on Whatman paper and covered with saran wrap at 75 degrees Celsius for 1.5 hours.

__**3. Results: **__

No plasmid control













[[image:uvvis.png width="429" height="284" caption="Figure 6: Absorption spectrum of Elution 1 [blue (Trial 2)  and black (Trial 1) and elution 2 [Red (Trial 1 and green (Trial 2)].  The absorbance measured at 280nm for Elution 1 was: 0.325  and 0.384, and for Elution 2: 0.011 and 0.00. Total yield as calculated by the Trial 2 Elution 1 at 280nm was 1.180mg"]]

A=βlc Average absorbance for Trials 1 and 2 for Elution 1: 0.355 c=A/(βl)=0.355/(38850*lcm)=9.138x10^-6//M// = 0.236mg/mL (What's the molecular weight you used to convert to mg/mL) yield=0.236mg/mL*5mL = 1.180mg protein.

Maximal wavelength calculations?





__**4. Discussion: **__

Discuss what happened during the expression process The investigation successfully demonstrated the validity of the protein characterization, purification and expression techniques employed. After lysozyme was used to break down larger cellular compartments and proteins and benzonase was used to break down the RNA and DNA so that these would become small enough to filter out of the Nickel matrix, the HIS tag system was used as a method of affinity chromatography. The 6x HIS tag that was added to the gbr22 protein bound more tightly to the Nickel than the other proteins found in the cell, and thus gbr22 was still embedded in the Nickel matrix after the wash solution was run through the column, and was only dislodged after the elution buffer of higher concentration competed for the Nickel binding sites. This result was demonstrated by the banding characterization seen in Figure 7. Sample 1 in lane 1 was the sample taken prior to purification and thus includes proteins of a huge variety of molecular weights. Sample 2 was taken after centrifuging and thus excludes the largest of molecules that fell out of solution, but Sample 2 still includes a large variety of proteins (Lane 3). Sample 3 was the waste flow after mixing the solution with Ni-NTA. The gbr22 band is theoretically lighter, due to the filtering and binding affinity tagging of the NIckel, but since the rest of the proteins are still in solution, the resolution is very low, and this effect cannot easily be seen. Sample 4 was the solution after the wash of low concentration imidazole. This dislodged the lightly bound (perhaps having a few HIS residues) proteins but kept the high affinity 6x HIS tagged gbr22 proteins bound to the Nickel and thus in the filter. Sample 5 was the sample taken after elution 1 and exhibits a very dark band at around 25 kDa compared to the Prestained Protein ladder standard. Sample 6 was the elution 2 sample and shows a significantly lighter band indicating that there was a very low concentration of gbr22 protein still lodged in the Ni matrix after elution 1.The size of the protein from the gel was qualitatively determined to be approximately 25,800g/mol while that found from the protein purification was 25,794.2g/mol. Thus, the gel electrophoresis successfully validated that the protein of interest was the one that was expressed and purified. However, the purity of the sample is not 100% as could be seen by Lane 6, Sample 5. There were a few light bands still present in the gel, along with the very dark band representing the gbr22 protein. It was approximated that the purity of the sample was 70% due to these apparent contaminant proteins of different MWs.

Some errors that appeared in this experiment included the contamination of the elution 1 solution. Seen as faint bands in lane 6, this error could be minimized by increasing the concentration of imidazole used to elute the column, or by running a greater volume of elution buffer through the column. Other errors included the drying of the gel. Figure 7 was taken prior to drying because the drying led to huge cracks through the gel. This may have been caused by the lack of wetting the gel prior to drying, or due to the unknown blue powder residue that can be seen at the top of Figure 7.

__**5. Conclusions: **__ The techniques used to express target proteins by overexpression in //E. Coli// followed by the use of Ni-NTA affinity chromatography column purification demonstrated successful production of gbr22 and purification as exhibited by the nanodrop spectroscopy in which the absorbance peak indicated a greater concentration of purple protein (gbr22) and by the electrophoresis where the darkest band after the elution was shown at the molecular weight that corresponded with gbr22. The techniques used demonstrated a relatively high purity purification of protein and could be used in future work to isolate proteins for X-ray Diffraction characterization as well as enzyme-binding assay analyses.

__**6. References: **__ <span style="font-family: Arial,Helvetica,sans-serif;">[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. <span style="font-family: Arial,Helvetica,sans-serif;">[2] Haghi F., Peerayeh S. N., Saidat S.D., Cloning, expression and purification of outer membrane protein PorA of Neisseria meningtidis serogroup B, // Journal of Infection in Developing Countries, //** 2011, ** 5(12), p856-862 [3] Zhou, W.; Merrick, B.; Morteza, K.; Tomer, K., Detection and sequencing of phosphopeptides affinity bound to immobilized metal ion beads by matrix-assisted laser desorption/ionization mass spectroscopy. //Journal of the American Society for Mass Spectrometry,// 11(4), 273-282