SPENCER+C

Could condense M&M more Use past tense! Missing images --elution 1 Improve captions--expand on images Discussion section not clearly marked, weak analysis

__ Title: __ __ Laboratory Expression, Purification, and Characterization of Gbr22 Protein __ __ Introduction: __ The production and purification of recombinant proteins has become more accessible due to the development of simple, commercially available systems [1]. It has also been aided by the existence of EMBL (European Molecular Biology Laboratory), a protein expression and purification facility that provides guidance, assistance, and information to research groups [2]. This has resulted in the amassing of protein data structures under the Protein Data Bank (PDB) for the public to see [1]. The uses of protein data structures are nearly limitless, as each protein can be implemented in a variety of ways itself. The technique of protein production and purification relies upon the initial overexpression of a recombinant protein within bacterial, yeast, insect, or human cells via plasmid insertion. Subsequent cell lysis, protein tagging, and protein analyzation methods such as gel electrophoresis are then used to purify and characterize the protein being investigated. Data retrieved can include approximations of the protein’s molecular weight, among other descriptors. The objective in this set of experiments was to overexpress, purify, and analyze a recombinant protein. The bacterial cells of //E. coli// were used as a transformation vector for the protein gbr22. Gbr22 is a fluorescent protein originally cloned from a coral from the Great Barrier Reef; because of its purple color, it can be tracked easily when purifying and characterizing. The hypothesis that the final protein samples tested would vary in observed purity compared to other individual protein samples in gel electrophoresis lanes was formulated before beginning the experiment. __ Materials & Methods: __ Transform competent bacterial cells (E. coli BL21 (DE3)) with the plasmid, pGEM-gbr22. Grow starter culture for bacterial colonies, then transfer starter culture to flasks for large culture protein expression via incubator. Harvest the purple media (cells) from the flasks. Create sample 1 from flask cells. Centrifuge the harvested cells in a conical tube; decant excess solution and weigh the leftover purple pellet at the bottom. Resuspend the cells in phosphate buffered saline 1x solution; add thawed stock lysozyme for a concentration of 1mg/ml, and vortex again. Lyse E. coli cells. Add cyanase to digest DNA/RNA within the mixture. Clarify the lysate by centrifuging and making sample 2. Isolate the soluble fraction using a P1000; dispose of cell debris. Syringe filter the lysate. Add Ni-NTA resin/buffer mix to the mixture. Incubate, and purify the protein from the Ni-NTA complex via column stripping. Use samples of flow through waste and wash as 3 & 4. Collect elution 1 (majority of purified protein) and elution 2 (dilute protein). Create sample 5 & 6 from elutions 1 & 2 respectively. Use Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) to collect absorbance spectra for gbr22 from Elution 1; measure at 280 nm. Determine the concentration of protein using 280 nm wavelength and Beer’s Law. Prepare the SDS-PAGE gel samples. Assemble the gel electrophoresis module (Model 26616, Thermo Scientific, Wilmington, DE), and pipette samples 1 through 6 into lanes of the gel. Run the electrophoresis module at 200 V for 25 minutes. Remove the gel, rinse it to remove excess SDS, mix the imperial protein stain and leave in a shaker for 1.5 hours until dark bands are visible. Destain the gel & rinse with water. Place a small KimWipe in the dish overnight in the shaker to remove background staining. The next day, dry the gel with Whatman filter paper. __ Results & Discussion: __ ** Figure 1. ** Day 1 bacterial plate with E. coli cells, 2/28/13, AMP + BL21(DE3), No plasmid added. ** Figure 2. ** Day 1 bacterial plate with E. coli cells, 2/28/13, AMP + BL21(DE3), pGEM-gbr22 (plasmid) added. In Figure 1 the bacterial plate with ampicillin but without plasmid insertion of pGEM-gbr22 is shown. This was run as a control in comparison to Figure 2, which shows the bacterial plate with ampicillin and with plasmid insertion of pGEM-gbr22. ** Figure 3. ** Day 2, 3/1/13, AMP + BL21(DE3), pGEM-gbr22 (plasmid added) shown with bacterial colonies. In Figure 3 the same plate from Figure 2 is shown after overnight transformation. Bacterial colonies (over two hundred) can be seen across the agar residue of the plate. ** Figure 4. ** Day 2, Evening, 3/1/13, (LB+AMP), BL21(DE3), pGEM-gbr22, after being left overnight in shaking incubator at 37 C and 200-350 rpm. Two flasks were made. In Figure 4, two flasks containing large cultures for the E. coli cells expressing gbr22 are shown. The flasks were left overnight in a shaking incubator. ** Figure 5. ** Day 3, 3/1/13, (LB+AMP), BL21(DE3), pGME0gbr22, purple cell pellet displayed after being centrifuged. Figure 5 shows the condensed pellet obtained from decanting the bacterial tube after centrifugation; the wet pellet weight was recorded as 0.32 grams. ** Figure 6. ** Elutions 1 & 2 shown in 15 ml conical tubes. In Figure 6 Elutions 1 & 2 are shown. Elution 1 was run through with a higher concentration of imidazole buffer, and purified more protein into the tube; Elution 2 utilized a lower concentration of imidazole buffer, and yielded a lower concentration of gbr22 in the tube; this accounts for the difference in colors. ** Figure 7. ** Trial 1 absorbance spectra for protein gbr22 at 280 nm using Nanodrop spectrophotometry. In Figure 7, the absorption spectra for gbr22 from Elution 1 at 280 nm using the Nanodrop spectrophotometer is shown. The protein yield was shown to be 0.17 mg/ml. The molecular weight of gbr22 was noted from online sources as 25794.2 grams [3]. Beer’s Law, A= εbc, and the final volume of Elution 1 were used to calculate the concentration of protein using the 280 nm wavelength, and the overall yield of gbr22 as follows: A= εbc (0.141) = (39,100)*(1)*(c) (0.141)/(38,850) = c c = (0.00000360613 mol/L) * (25794.2 grams)
 * c = 0.09301723844 mg/ml **

280 nm: [0.09301723844 mg/ml] * [4.6 ml] = **0.42787929682 mg gbr22**

The calculated concentration of gbr22 within Elution 1 was 0.09301723844 mg/ml, and using the measured volume of Elution 1, the yield of gbr22 was shown to be 0.42787929682 mg. ** Figure 8. ** Gel after final destaining and drying process. The lane for Elution 1 is third most to the right. In Figure 8 gel lanes are shown after the drying process. Sample 1 (bacterial cells before centrifugation) is to the right and left of the multicolored lane (molecular standards lane) for precision. This lane is almost entirely colored as it contained the cellular lysate components. Lysozyme was used to break down the bacterial cell walls (sample 1), and cyanase was used to degrade any DNA/RNA within the solution. Sample 2 (bacterial cells with lysate solution) is shown to the right, with sample 3 (flow through waste from 1xPBS rinse) shown to the right of sample 2. The next lane is nearly empty and had to be redone in the next lane as sample 4 (wash solution). The lane with the clear dark band third from the right contained sample 5, aka Elution 1, and the lane to the right of that contained sample 6, aka Elution 2. Sample contents were successively purified from samples 1 through 6, supporting the decrease in purple color as the samples continue to the right direction. The elution buffer had less imidazole than the wash buffer; the increased concentration of imidazole in the wash buffer purified more of the protein gbr22 by separating from the Ni-NTA/HIS tag complex. ** Figure 9. ** Image of molecular weight ladder from Bio-Rad [4]. When comparing the band from sample 5’s lane to the molecular weight standards chart (Figure 9), the molecular weight of the purified protein was estimated to be 28.5 kDa. The size determined in the protein purification lab was 25.7942 kilograms; the estimated size based upon the protein band is then shown to be accurate. The purity of sample 5 was very high, due to the absence of other protein bands within the lane, and could be estimated as 90% (see Figure 8). Sources of error in this lab were numerous; one main source of error included the fact that the wash and elution buffers were made individually, and therefore were subject to slightly varied or incorrect concentrations. Other sources of error include the subjective nature of reading Figure 9 for comparison to the band from Sample 5 (Elution 1) in Figure 8 (the gel lanes). __ Conclusions: __ This experiment involved overexpression of the protein gbr22 in bacterial E. coli cells, and subsequent purification and characterization of the protein samples. Nanodrop spectrophotometry was used to measure the protein’s absorbance spectra at 280 nm; this data was used to calculate the concentration and total yield of gbr22. Gel electrophoresis was run on a variety of samples to note in which steps the protein became the most pure and to estimate the protein’s size. Key results included the concentration and yield of gbr22, as well as the indication that gbr22 was very pure within elution 1. When comparing this purity to other gel lanes, the original hypothesis that different lanes would show different purities of gbr22 was supported. Future applications of this experiment include producing and purifying other proteins necessary for biomedical research or drug therapy. Other applications include repeating the experiment with different methodologies to maximize protein yield from bacterial expression. __ References: __
 * 1) Protein production and purification. //Nat Methods// **2008**, //5 (2)//, 135 – 146.
 * 2) European Molecular Biology Laboratory. Protein Expression and Purification Core Facility. http://www.embl.de/pepcore/pepcore_services/index.html (accessed April 16, 2013).
 * 3) ExPASy: Bioinformatics Research Portal. Protparam Tool. http://ca.expasy.org/tools/protparam.html (accessed April 17, 2013).
 * 4) Bio-Rad. Precision Plus Protein Prestained Standards. http://www.bio-rad.com/prd/en/US/adirect/biorad?ts=1&cmd=BRCatgProductDetail&vertical=LSR&catID=a7b0f9ce-e080-4b51-ab99-4cded66497c1 (accessed April 16, 2013).