SHIVANI+B

Intro should be more background info on theory or on the technique than restating methods Reduce M&M to most critical details Discussion not very profound, restated some methods Improve references __ Title: __ Why so purple? Gbr22 Expression, Purification, and Characterization

__ Introduction: __ Recombinant proteins are used widely in the biological sciences. In recent years, there has been a significant increase in the techniques and products used in the amplification and purification of recombinant proteins. Tens of thousands of proteins have been purified and added to large databases. Restriction enzymes can be used to clone genes into different expression vectors. [1] Cloning strategies consist of gene amplification by PCR, sequence analysis, and cloning into expression vectors. //E. coli// are commonly used in protein expression. Recombinant //E. coli// colonies can be grown to speed up protein production. Protein purification techniques involve extraction and clarification, which results in the preparation of a clarified sample of the soluble protein and the removal of unwanted particulate matter. Chromatography techniques are then utilized to increase purity of the sample. [2] The objective of this lab was to express, purify, and characterize gbr22, a fluorescent protein that was originally cloned from a choral from the Great Barrier Reef. It was hypothesized that successful expression and purification of the gbr22 protein in // E. coli // cells would be demonstrated through the presence of a band in the gel and through the purple coloration of the cell cultures and protein solutions.

__ Materials & Methods: __ Competent //E. coli// bacteria ( New England BioLabs, Ipswich, MA ) were used to over express the protein of interest, gbr22. The plasmid pGEM-gbr22, carrying a gene for ampicillin resistance, was inserted into //E. coli// BL21 host bacteria through bacterial transformation technique (30 minutes on ice, heat shock in 42 ̊ C for 45 seconds, ice for 2 minutes) and grown on LB-agar plates with ampicillin (a negative control plate was made with untransformed bacteria to confirm sterile technique). Plates were incubated at 37 ̊ C overnight. A starter culture was grown and incubated at 37 ̊ C for 8 hours in LB supplemented by ampicillin from one of the colonies on the LB-agar plate with transformed bacteria. A large culture was then grown from the starter culture and incubated at 37 ̊ C for 16-24 hours in LB-ampicillin solution. The transformed cells were harvested by centrifuging a sample of the large culture and saving the pellet full of cells. Cells were re-suspended in PBS solution and lysozyme was added to break the cell walls. Cells were then stored in a -20 ̊ C freezer.

Benzonase was later added to the thawed cells, and the resulting lysate was then centrifuged and the supernatant with the soluble proteins was saved. Ni-NTA chromatography was used to purify the gbr22 protein. PBS buffer solution was first flowed through the column and collected. The column was then washed to dislodged weakly bound proteins with 20 mM Imidizole in PBS solution (wash step). A 250 mM Imidazole solution was then used to elute the bound protein (elution 1 and elution 2). The concentration of the final purified protein was estimated using a Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE).

Samples were prepared for SDS-PAGE with 6x loading buffer (glycerol, DTT, SDS, bromophenol blue). Sample 1 (cell fraction), sample 2 (soluble fraction), sample 3 (flow through from centrifugation), sample 4 (wash), sample 5 (elution 1), and sample 6 (elution 2) were loaded in the gel lanes along with a Molecular Weight ladder. The gel was run, stained with Imperial protein stain, destained, and dried.

__ Results: __













Extinction coefficient at 280 nm = 38850 Molecular Weight of gbr22 = 25794.2 Beer's Law: A = εbc 0.158 = 38850 c C = 0.0000041 mol/L

((25794.2 g)/(1 mol))((0.0000041 mol)/(1 L))((1 L)/(1000 mL))((100 mg)/(1 g)) =0.1049 mg/mL





__ Discussion: __ Competent //E. coli// bacterial cells were transformed to express the gbr22 purple protein. Since the pGEM-gbr22 plasmid that was incorporated into the bacterial cells contained a gene for ampicillin resistance, colonies growing on the ampicillin positive agar plate (see Fig. 1a.) indicate that these colonies were successfully transformed. Figure 1b demonstrates the inability of untransformed bacteria to grow due to the lack of an ampicillin resistance gene, serving as the negative control. A large culture was grown from a single colony of transformed bacteria, and the purple color of the large culture (see Fig. 2) indicates that the cells were successfully expressing the purple protein. After centrifuging a sample from the large culture and removing the supernatant, the pellet, weighing 0.73 g, contained the cells transformed with the purple plasmid.

After the protein was successfully expressed, the protein purification process involved the use of lysozyme to break the cell walls of the bacterial cells to allow release of the proteins inside the cells. Benzonase was then used to digest the DNA and RNA in the mixture so that protein was left in the solution. After centrifugation, the supernatant contained the soluble proteins (including gbr22) from the cell (cell debris and DNA/RNA was discarded as the pellet). Ni-NTA chromatography allowed the separation and purification of the gbr22 protein. The gbr22 protein was modified to have 6 histidine residues at the C-terminus of the protein. These histidine residues, or HIS tags, were used to separate the gbr22 protein from other soluble proteins in the cell due to the histidine affinity to Ni-NTA. Imidazole, which competes with histidine residues for metal binding, was used in low concentration first to wash other soluble proteins away, then in higher concentration twice to release the tightly bound gbr22 protein (elution 1 and elution 2).

Sample 1 in the SDS-PAGE gel contained the cell fraction, which consisted of the gbr22 protein inside the bacterial cell along with all other cellular proteins, thus the sample 1 band appeared the darkest. Sample 2 contained the soluble fraction, after the cell debris and DNA/RNA were digested and removed. This lane appeared dark as well since many proteins in addition to gbr22 were present. Sample 3 consisted of the flow through during Ni-NTA chromatography, which contained all proteins in the soluble fraction that did not bind to the metal, thus causing the lane to be dark. Sample 4 contained the contents after the wash step of column chromatography, which caused all loosely bound soluble proteins to flow through the column. This caused the entire lane to be dark again, but less so than the previous lanes (since fewer proteins were present at this stage). Sample 5 was collected after the elution buffer was run through the column once and contained the gbr22 protein, thus having one dark band in the lane. Sample 6 was collected after the elution buffer was run through a second time, containing the gbr22 protein again and thus having one dark band (but lighter than the sample lane, since most of the gbr22 protein had already been eluted).

As seen in Figure 6, there were two light bands in addition to the one corresponding to gbr22 in lanes 6,7,9, and 10. These bands indicate that the purity of the samples was not 100%, but rather approximately 40%. Using the molecular weight ladder, the size of the gbr22 protein in the gel was estimated to be approximately 25 kDa. This compares well to the protein size that was determined earlier in the experiment as 25794.2 g/mol.

Contamination may have been a large source of error in this lab, as demonstrated as the extra bands that appeared in the gel. Contamination errors could have arisen in the bacterial transformation steps through improper sterile technique.

__ Conclusions: __ The gbr22 protein was expressed in competent //E. coli// bacterial cells through transformation and uptake of the pGEM-gbr22 plasmid. After the cells were grown to express the protein, the gbr22 protein was purified through Ni-NTA column chromatography. Protein characterization was subsequently conducted through an SDS-PAGE run, and the molecular weight of the protein was determined. In the future, protein expression and purification techniques can allow researchers to produce and analyze novel proteins. These proteins could be used as drug-targets or may serve other functions in biological research.

__ References: __

====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).====
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