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Missing nanodrop elution 2 __ Title: __ **Purifying ****Pulchritudinous Purple Protein: Expression, Purification, and Characterization of pGEM-gbr22 **

__ Introduction: __

Studying proteins and their functions increases our biochemical understanding of cells and organisms. Techniques to isolate, amplify, and purify proteins have increased in recent years [1]. Tens of thousands of proteins have been targeted and purified in the last decade. Proteins of interest must first be obtained from a natural source, cloned into different expression vectors, and expressed in a host organism (such as //E. coli//) [1]. Proteins must be purified by preparing the bacterial lysate and performing chromatography to elute other proteins and weakly bound contaminants [2]. A particular chromatography technique (gel filtration, ion-exchange, affinity chromatography, etc.) will be used depending on the properties of the protein of interest (i.e. size, hydrophobicity). Characterization techniques including inspection of chromatograms, SDS-PAGE analysis, and UV-Vis absorption spectroscopy confirm the identity, purity, and quantity of the protein [2].

The objectives of this investigation were to overexpress the recombinant pGEM-gbr22 protein in competent //E. coli// BL21(DE3) cells, purify the protein using affinity chromatography, and characterize gbr22 with an SDS-PAGE analysis. It was hypothesized that with proper technique, successful expression, purification, and characterization of only the gbr22 protein would be illustrated with purple coloration of cell cultures and with the presence of one band of appropriate molecular weight after gel electrophoresis.

__ Materials & Methods: __

//Protein Expression //

Competent //E. coli// BL21(DE3) bacterial cells (New England Biolabs, Ipswich, MA) were transformed with the pGEM-gbr22 plasmid. Plasmid was inserted into 25 uL of host cells through centrifugation, waiting on ice for 30 minutes, heat shocking tubes in 42 ̊C water bath for 45 seconds, waiting 2 minutes on ice, adding 200 uL of SOC media, and shaking in the incubator for 30 minutes at 37 ̊C at 250 rpm. 50 uL of bacteria/SOC mixture was grown on LB-agar + ampicillin and one control plate (no DNA) and incubated overnight at 37 ̊C. A starter culture was grown in LB with 100 ug/mL ampicillin and one transformed bacterial colony with aseptic technique and incubated at 37 ̊C for 8 hours. Large culture for overnight expression was made by incubating at 37 ̊C for 16-24 hours after adding 25 mL fresh LB, ampicillin, and 0.625 mL of the starter culture. Transformed cells were harvested by centrifuging the large culture, saving the pellet, re-suspending cells in PBS solution + lysozyme, and storing in the -20 ̊C freezer.

//Protein Purification //

Transformed cells were thawed and 2 uL of benzonase was added. Lysate was centrifuged; supernatant with soluble proteins was saved. Supernatant was filtered through 0.45 um PES syringe filter, and run through an Econo column with 0.5 mL of Ni-NTA resin/buffer. S3 was collected, and 5 mL of wash elution was added to column (1x PBS + 20 mM imidazole). S4 was acquired, and 5 mL of elution was added twice (1x PBS + 250 mM imidazole). S5 and S6 were collected. Nanodrop spectrophotometer ( Thermo Scientific, Wilmington, DE) at λ = 280 and 574 nm was used to estimate quantity of gbr22 protein.

//Protein Characterization //

SDS-PAGE analysis was used by preparing samples with 6x loading buffer (DTT, SDS, bromophenol blue, glycerol). S1-S6 (student 1) and S4-S6 (student 2) were loaded into gel lanes with the MW ladder. 4-20% Tris-glycine gel was run, stained with Imperial protein stain, destained, and dried for analysis.

__ Results: __

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Beer's law ( A= e bc) calculations to determine yield of pGEM-gbr22 protein:

Concentration at 280 nm: Average A: (0.288 + 0.334) / 2 = 0.311 C = 0.311/[(38850 M-1 cm-1)(1 cm)]= 8.01-6 mol/L (25,794.2 g/mol) = 0.206 mg/mL

Concentration at 574 nm: Average A: (0.33 + 0.31) / 2 = 0.32 C = (0.32)/[(118300 M-1 cm-1)(1 cm)]= 2.70-6 mol/L (25,794.2 g/mol) = 0.0698 mg/mL



__ Discussion: __

Competent //E. coli// BL21(DE3) cells were successfully transformed with the pGEM-gbr22 plasmid, as bacterial growth occurred on the LB+amp plate (Figure 1a). The plasmid contains a gene for ampicillin resistance which enabled the bacteria to grow on the plate. In addition, the absence of bacterial growth on the control plate validates the use of aseptic technique (Figure 1b). Purple coloration of the large culture of BL21(DE3) bacterial cells from a single colony indicates successful transformation of //E. coli// with plasmid DNA pGEM-gbr22 (Figure 2). Figure 3 illustrates the 0.38 g wet pellet acquired from centrifugation of large BL21(DE3) bacteria culture transformed with plasmid DNA pGEM-gbr22.

Once the protein of interest was overexpressed in //E. coli,// the purification process included lysozyme to break the cell walls of the bacteria so proteins could be released. Benzonase was used to digest DNA and RNA, which would leave behind proteins. Ni-NTA chromatography separated and purified the gbr22 protein. This was accomplished by the HIS tag system, as the gbr22 protein was modified with 6 HIS residues at the C-terminus. The HIS affinity to Ni-NTA allowed the gbr22 protein to be retained, while other soluble proteins flowed out the column. The wash buffer included a low concentration of imidazole, which competes with the HIS residues for metal binding. In contrast, the elution buffer contained a higher concentration of imidazole, to release the gbr22 protein.

The SDS-PAGE analysis included samples 1-6 in the wells of the gel. The contents of the sample comprised of: S1 (cell lysate – gbr22 protein with other soluble proteins), S2 (soluble fraction – after cell debris and DNA/RNA were removed), S3 (flow through during affinity chromatography), S4 (wash – after adding 20 mM imidazole), S5 (elution 1 – after adding 250 mM imidazole), and S6 (elution 2). Since reagents increased in both concentration (imidazole) and selectivity for the gbr22 protein from S1-S6, it is expected that the # of protein bands decrease from S1-S6. The dried gel (Figure 7) illustrates this pattern. The size of the protein from purification was determined to be 25794.2 g/mol, whereas the results from gel electrophoresis indicated a size of 25 kDa (Figure 8). These values are relatively close and indicate that the characterization of the gbr22 protein was successful. S5 data from Figure 7 shows that there were about 2 other protein bands present in the lane for both students. S.Z. had 1 other band of equal intensity, and thus the purity of the sample was approximately 50%.

Sources of error in this investigation include contamination of samples during transformation (improper aseptic technique), purification, or characterization (improper gel loading technique). This is illustrated by the extra bands present in the gel. In addition, all of the gbr22 protein may not have extracted/purified from the chromatography column, thus resulting in a lower concentration or yield.

__ Conclusions: __

<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">Competent //E. coli// BL21(DE3) cells were successfully transformed with the pGEM-gbr22 plasmid and purified using Ni-NTA affinity chromatography, indicated by the purple coloration of cultures and solutions, as well as the absorbance peak from the Nanodrop spectrophotometer. Characterization with SDS-PAGE analysis illustrated a 50% purity of the sample, and the highest intensity band at the location corresponding with the molecular weight of the gbr22 protein. These techniques can be utilized in future VDS research to isolate, express, purify, and characterize proteins of interest that can be studied and analyzed.

__ References: __

1. <span style="font-family: 'Times New Roman','serif'; font-size: 16px;">Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards,A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.;Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.;Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.;Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.;Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C.; Consortium, S. G.; Consortium, C. S. G.; Consortium, N. S. G., Protein production and purification. //Nat Methods// **2008**, 5 (2), 135-46.

<span style="font-family: 'Times New Roman','serif'; font-size: 16px;">2. European Molecular Biology Laboratory. Protein Expression and Purification Core Facility. [|http://www.embl.de/pepcore/pepcore_services/protein_purification/purification/index.] (accessed April 17, 2013).