KAVYA+K

 Protein Purity


 * __ Introduction: __**


 * Protein in bacteria can be overexpressed by cloning a gene and placing the gene into a plasmid, a circular piece of DNA that exists apart from the** (finish sentence here) **. Bacterial plasmids carry information that renders the bacteria (BL21 (DE3) in this case) resistant to antibiotics. In this lab, pGEM-gbr22 was obtained because it was easy to follow the purification of the protein because it was purple in color and also contained a gene for ampicillin resistance. When transforming the competent bacterial cells, plasmid was placed only into one tube so some bacterial colonies could take up this plasmid while others may not. Ampicillin ensured that only the bacteria that took up the plasmid survived (have resistance to the ampicillin).** (I feel like this should be later in the report)


 * Protein purification allows for soluble proteins from bacterial cells to be purified and prepared for characterization. This includes several processes though which protein is separated from the non-protein part through centrifuging, and then from other proteins by utilizing the His-tag system [2,3]. However, there is not one single strategy that is efficient in purifying all proteins, because each protein is unique [3]. For example, the affinity tag can be removed to minimize non-native sequences in the recombinant protein. In this lab, the objective was to purpose of this lab is to overexpress a recombinant protein in bacteria, purify this protein from other proteins, and then characterize to obtain a purity of the sample. Through proper transformation and purification techniques, the electrophoresis gel should only display one band implying that 1 protein was present in the sample.** (Intro could be overall a bit stronger and talk more about the theory behind expression, purification, and characterization)


 * __ Materials & Methods: __**


 * The bacterial cells (25µl) (BL21 (DE3)) (NEB New England Biolabs) were transformed by adding plasmid DNA (pGEM-gbr22/300ng) to one tube, heat shocked and plated. After incubation overnight** (conditions?) **, a starter culture was grown by adding 100 µg/ml of ampicillin and 1 bacterial colony to a tube and incubating for 8 hours. 0.625ml of the starter culture, 25ml of LB, and 5µl of ampicillin were incubated overnight** (conditions?) ** . The cells were harvested (sample1), resuspended in 2.5ml of 1X PBS and lysozyme was added. ** (Be more specific about how much lysozyme, etc)


 * 1 µl of Cyanase was added and the lysate was centrifuged (sample 2). The soluble fraction was isolated and syringe filtered through a .45µm Mille filter. 0.5ml of NI-NTA resin/buffer mix was added to the protein and incubated for 15 minutes. Resin/buffer and was added to the Bio-Rad Eco no column and flowed into “waste” (sample 3) and 5ml of 20mM imidazole into “wash” (sample 4). The protein was eluted with 5ml of 250mM imidazole into tube “elution 1” (sample5) and again as “elution 2” (sample 6). An absorbance reading was taken at 280nm and 540nm for elutions 1 and 2.**


 * Sample 1 was centrifuged and resuspended in water and samples 1-6 were heat blocked. For the gel electrophoresis, molecular weight standards (Pageruler, Prestained, 26616, Thermoscientific) were placed into well 1, and samples 1-6 and 4-5 (of the second partner) respectively into the remaining cells. After 25 minutes, the wet gel is placed in orbital shaker with imperial stain. The next day, the gel is dried by placing it on a gradient cycle for 1.5 hours.** (Explain the volts used in SDS-PAGE, and things like the temperature used to dry gel)

__** Results **__




 * Figure 1. Day 2 (2/28/13) Agar plates with BL21 (DE3), plasmid vector (pGEM-gbr22), //E.coli.//**
 * Bacterial colonies displayed as white dots.** (Ampicillin grown at what conditions?)



(ampicillin, grown at what conditions?)
 * Figure 2. Day 2 (2/28/13)**
 * Agar plates with BL21 (DE3), No DNA Control, //E.coli//.**
 * Only 1 single bacterial colony displayed as white dot.**



(ampicillin, grown at what conditions?)
 * Figure 3. Day 2 (2/28/13)**
 * Fun Plate (coughing), and no antibiotic**
 * No bacterial growth visible**




 * Figure 4. Day 3 (3/1/13)**
 * Purple culture in flask with BL21 (DE3), plasmid vector (pGEM-gbr22),**
 * and LB + Amp.** (what was done to get here? Ex: shaking incubator)


 * Figure 5. Day 3 (3/1/13)**
 * Purple Cell pellet (.36 g) in 15ml conical tube; with BL21 (DE3), plasmid vector (pGEM-gbr22),**
 * and LB + Amp.**


 * Figure 6. Elution 1: purple/with most protein with**
 * with 1x PBS with 250 mM imidazole and Elution 2 (clear) with 1x PBS with 250 mM imidazole.** (imidazole isn't the only thing in the elution buffer)


 * Figure 7. Absorbance Spectrum of Elution 1 at 280nm (n=1) Trial 1 (yield = 1.382 mg)**


 * Figure 8 (left). Figure 9 (right).**


 * Figure 8 (left) and 9 (Right) Wet Gel/ Dry Gel (Gradient Cycle for 1.5 hours at 75 degrees Celsius)**
 * Well 1: Marker**
 * Well 2: Cell Lysate**
 * Well 3: Soluble Fraction**
 * Well 4: Flow through (waste = Lyzozyme, LB, PBS,etc.)**
 * Well 5: Wash (20mM imidazole)**
 * Well 6: Elution 1**
 * Well 7: Elution 2**
 * Well 8 (sample 4/2nd partner): Wash (20mM imidazole)**
 * Well 9 (sample 5/2nd partner): Elution 1**
 * Well 10 (sample 6/2nd partner): Elution 2**
 * Figure 10.**
 * Molecular Weight Standard (Gel vs. Western Blot)**
 * Pagemore Prestained Protein Ladder, 26616, Thermoscientific**


 * Calculations:**
 * A = εcb**
 * 574nm**
 * .85 = 118300 (1)[c]**
 * [c] = 7.185x10^-6mol/L(25794.24g/mol)=.185mg/ml**
 * yield = .185mg/ml(5ml) = .926mg**


 * 280nm**
 * .4165 = 38850 (1)[c]**
 * [c] = 1.07x10^-5mol/L(25794.24g/mol)=.276mg/ml**
 * yield = .276mg/ml(5ml) = 1.382mg**

__** Discussion: **__


 * To prepare for characterization Sodium Dodecyl Sulfate separates the protein and applies a charge, which allowed the proteins to move towards the positive end. The distance of the migration was only related to the mass of the protein [1].**
 * In sample 1, the protein was present because the cell lysate contained the protein. When purifying the protein, the lysozyme digested the cell walls and the Cyanase digested the DNA and RNA. In sample 2, the soluble fraction contained the protein because the protein was originally in the cytoplasmic portion of the cell (band present). After filtration, the Ni-NTA resin buffer was added so it could bind to the protein, and the “waste” (sample 3) contained the flow through (Lysozyme, LB, PBS, etc). In sample 3, flow through, the protein should not have been present because the histidine tag attached to the protein was also attached to the Nickel which was attached to beads that prevented the protein from flowing through. There was no protein and thus a band should not have been present.**


 * A low concentration of imidazole (20mM) knocked off any loosely bound protein into the wash (sample 4). Ideally, there should not have been a band present in this sample; however some loosely bound protein may have flown through. A high concentration of imidazole (250mM) knocked off the rest of the protein into “Elution 1” (sample 5) because the histidine residues are able to attach to the imidazole** (imidazole competes with the Ni-NTA resin) **. Thus Elution 1 contains more protein than Elution 2 and the wash. Sample 5 (elution 1) should have only contained 1 band in the dried gel because only 1 protein was present after eluting with the strong concentration of imidazole (250 mM). For sample 6, any left over protein was contained in elution 2. This band appeared lighter because there was less protein. Thus the overall purify was about 33%, which is low. The size of the protein from the gel was 25kda while the estimated weight was 25.7942kda.**


 * Sources of error include sample 5 (elution 1), which was to include one band. 3 bands or 3 proteins may have been present because some other plasmids may have been introduced into the bacteria. Also, the flow through and wash (samples 3 and 4) should not have contained only protein bands** (Yes they should have contained all the other junk proteins E. coli made **). This might have occurred because some of the protein accidentally flowed through just as in the wash. Overall, the plasmid may have been contaminated.**

(Need to talk more about theory here. About difference in imidazole concentrations, what lysozyme does, etc.)

__** Conclusions: **__


 * Overall, the gel electrophoresis displayed that the proteins had not been purified properly because sample 5 displayed 3 proteins as opposed to 1. Thus the purity of the sample was low at about only 33 because some contamination of the plasmid may have occurred. This method of transforming the bacteria, purifying, and then characterization will allow for certain proteins to be visible. The chemical structures of these proteins can be analyzed to help determine the role in protein affinity.** (What are the next steps we want to do with the protein?)

__** References: **__


 * 1. Aebersold, R.; Leavitt, J., Sequence analysis of proteins separated by polyacrylamide gel electrophoresis: towards an integrated protein database. //Electrophoresis// 1990, //11// (7), 517-27.**


 * 2. Halavaty, A. S.; Kim, Y.; Minasov, G.; Shuvalova, L.; Dubrovska, I.; Winsor, J.; Zhou, M.; Onopriyenko, O.; Skarina, T.; Papazisi, L.; Kwon, K.; Peterson, S. N.; Joachimiak, A.; Savchenko, A.; Anderson, W. F., Structural characterization and comparison of three acyl-carrier-protein synthases from pathogenic bacteria. //Acta Crystallogr D Biol Crystallogr// 2012, //68// (Pt 10), 1359-70.**


 * 3. Structural Genomics Consortium. China Structural Genomics Consortium. Northeast Structural Genomics Consortium. Gräslund S, Nordlund P, Weigelt J. Protein production and purification. Nat Methods. 2008, 25 :135–46.**