NICOLE+W

Intro should be more background info on theory or on the technique than restating methods Reduce M&M to most critical details Improve calculations Discussion not very profound, weak analysis Flow of discussion hard to follow Missing error analysis __The process of expressing, purifying and characterizing bacterial protein__

__Introduction:__

Recombinant proteins are used for many research reasons, but every protein has an unique process of purifying that protein [1]. The process of expressing most protein involves obtaining cDNA, using LIC to clone, using T7 RNA polymerase, induce at a low temperature, solubilize and purify and gel filtration [1]. In order to express protein a single colony must be picked to make a started culture. Then there are tags utilized during the process of purifying proteins to speed it up but they don’t denature the protein. Later gel filtration chromatography is used to increase the purity of the separated protein samples [3]. One example of successfully expressing protein is the protein GAGE which can treat a wide range of cancer patients with small side effects [2].

The overall purpose of these three labs was to characterize the bacterial protein to determine its' molecular weight and concentration. The point of the protein expression lab was to express recombinant protein with DNA plasmid and harvest cells to freeze for later. The point of the purifying lab was purify the protein that was previously expressed by centrifuging, affinity tag and Ni-NTA resin. The point of the characterization lab was to estimate the molecular weight of the protein for the samples using gel electrophoresis (SDS-PAGE).

If bacterial protein is expressed using DNA plasmid and the same protein is purified by centrifuging then by utilizing gel electrophoresis the concentration and molecular weight of that protein can be determined.

__Methods and Materials:__

 Goggles, coat, gloves, ice bucket, water bath, gas burner, 14&10 ml round-bottom tubes, incubator, colirollers, Agar plates(one w/o antibiotic), competent cells, plasmid DNA, LB, SOC, pipette, pipette tips, gas burner, Ampicillin, Imidazole, PBS, centrifuge tubes,conical tubes, Bio-rad Econo chromatography column, ring stand, clamps, Ni-NTA resin, Cyanase, Heat block, mini-PROTEAN electrophoresis tank, power supply, leads, TGS running buffer, Bio-Rad precast polyacrylamide gel, plastic container and imperial protein stain.  Expression: Use busen burner when handling bacteria. Add 25uL of bacteria and 1.5965uL of plasmid to the DNA tube wait 30 minutes then heat shock. Add 200uL of SOC media then shake in incubator 30 minutes.Put 5-6 colirolers and 50uL mixture on plate then mix. Store in incubator overnight. Later add .01mL Ampicilin, 5mL of LB and single colony from plate and put in tube. Transfer 25mL of LB to 125mL Erlenmeyer flask, add 50mL of Amp then .0625mL of culture and shake incubate for 16-24 hours. Centrifuge bacteria for 10 minutes then weigh pellet. Add 2.5mL of 1XPBS and 50 uL lysozyme then freeze.  Purification: thaw pellet then add 1uL of cyanase. Distribute lysate into tubes and centrifuge for 20 minutes. Then take 50uL of supernant (sample2). With just the pellet syringe filter it then add .05mL Ni-NTA. Transfer this to the column and collect clear flow-through(sample3). Wash with 5mL of wash buffer(sample4). Wash with 5mL of elution buffer and repeat(sample5&6). Use nanodrop spectrophotometer to determine absorbance.  Characterization: Centrifuge sample1 for 5 minutes then remove liquid and add 40uL of loading buffer. Add loading buffer to samples 2-6 then centrifuge all samples for 5 minutes. Then add 7uL of each sample to the gel and let run for 25 minutes. Remove gel from plate in dish of nanopure place on orbital shaker and wash gel three times. Let shake for 1-1.5hours then rise twice again and add KimWipe to shake overnight. Cover gel with Whatman paper and Saran wrap the dry for 1.5 hours.

__Results:__

__ Images from Protein Expression Lab: __ Figure 1: Day 2 (morning) bacterial/dna plate with pGEM-gbr22 and BL21 (DE3) on Amp plate after overnight incubation at 37 ° C. One colony was taken. Figure 2: Day 2 (morning) control plate with no DNA on Amp plate after overnight incubation at 37° C. Figure 3: Day 2 (morning) fun plate bacteria collected for the sink on an Agar plate with no DNA and no Amp after overnight incubation at 37 ° C.  Figure 4: Purple culture bacteria on day three after 16-24 hours of incubation at 3 7 ° C; contains LB, Amp, BL21(DE3) and DNA pGEM-gbr22. Figure 5: Pellet formed from bacteria solution after centrifuged for 10 minutes at 5,000rpm at 4°C on day 3. The pellet weighed 0.23 grams.

__Images from Protein Purification Lab:__

Figure 6: Elution one tube containing expressed protein, and 5 mL of 1xPBS and 250 nM imidazole. Figure 7: Elution two containing less expressed protein than before, and 5 mL of 1xPBS and 250 nM imidazole. Figure 8: Absorbance graph of Elution one (trial one) at the wavelength of 280 nm using the Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE). Absorbance is 0.385. Figure 9: Absorbance graph of Elution one (trial two) at the wavelength of 280 nm using the Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE). Absorbance is 0.392. Figure 10: Absorbance graph of Elution one (trial one) at the maximum wavelength of 574 nm using the Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE). Absorbance is 0.04. Figure 11: Absorbance graph of Elution one (trial two) at the maxiumum wavelength of 574 nm using the Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE). Absorbance is 0.041.

Beer's Law Calculations: A=ebc Absorbance at wavelength of 280 nm: Trail one=.385; Trail two=.392 these values are A in the equation. Extinction coefficient from protein sequence 38850 this is e. C is being calculated then this value is then multiplied by the molecular weight of gbr22 (2579.42 g/mol) and (1000mg/1000mL) to give the proper concentration in mg/mL. The same process is repeated at the wavelength of 574 nm.

__Images from Protein Characterization Lab:__ Figure12: Gel electrophoresis (SDS-PAGE) after solutions from 1-6 were added then shaken overnight in the shaken incubator before the gel was dried.  Figure 13: Gel electrophoresis (SDS-PAGE) after dried in the vacuum for 1.5 hours. Ladder, cell lysate, SOL, flow through, was, elution 1 and elution 2 are labeled on the gel. Figure 14: PageRuler Prestained Protein Ladder (product #26616) used as the ladder in the gel electrophoresis. Molecular weight 25 kDa.

__ Discussion: __

Q1: Breaks open bacterial cells by digesting cell wall, catalyzer hydrolysis and effective against Gram positive bacterial cells. Cynase will reduce the viscosity by digesting the DNA/RNA and gets rid of the DNA/RNA that is not needed. Q2: The six histidine resudes attach at the C-terminues (end of the protein) to separate the protein from other things in the solution. Nickel metal bings to the Histine residues not allowing the protein to go through the column until the elution buffer is used. <span style="font-family: Arial,sans-serif; font-size: 10pt;">Q3: Sample one was cell lysate so protein was present. Sample two was SOL and a small amount of protein was present. Sample three was a flow through, no protein (the protein being worked with) should have been present. Sample four was wash buffer and no protein should have been present just the other substances in the solution. Sample five was elution one and should only have the protein and sample six should have whatever protein that was left in the column. <span style="font-family: Arial,sans-serif; font-size: 10pt;">Q4: The was buffer is supposed to remove everything else but the protein being worked with. While the elution buffer has imidazole that competes with the histidines to elute the protein off the beads. <span style="font-family: Arial,sans-serif; font-size: 10pt;">Q5:The size of the protein determined for the gel is 25 kDa which is very similar to the size determined in the protein purification lab. <span style="font-family: Arial,sans-serif; font-size: 10pt;">Q6: The purity of the protein is 30% because of the other substances that were present in the solution along with the protein. <span style="font-family: Arial,sans-serif; font-size: 10pt;">Q(handout): The protein stayed in the filter until the elution buffer was used. The 6xHis tag and the protein stuck to the nickel. The protein sticks to the Ni-NTA beads and the nickel so it doesn’t flow through the filter until elution buffer.

<span style="font-family: Arial,sans-serif; font-size: 10pt;">Sources of error: In the expression lab the busen burner may not have been enough to keep the area sterile and clean from other bacteria. In the purification lab the elution buffer may not have worked the first time so not all of the protein came out in sample five and not all of the protein and other substances came out before elution buffer was used causing the absorbance readings to be off. Overall the major source of error in the experiment was that the plasmid was made with more than one protein so another protein besides the one being worked with was expressed and seen in the gel. This error could have been prevented by checking the plasmid first to make sure there was only the protein being worked with in it.

__ Conclusions: __

In the expression lab protein grew on the DNA plate and the starter culture turned purple. In the purification lab elution one was more purple than elution two showing that there was more protein in elution one as it should have been. Figures 8&9 and 10&11 are extremely similar to each other showing that nanodrop spectrophotometer calculated the absorbances accurately and precisely. The conecentration calculated at wavelength=280 nm and the max wavelength were close to eachother showing the protein was properly purified. But in the protein characterization lab the gel showed that there was another protein present as seen as the dark purple line near the very bottom of the gel which was not supposed to be there. The molecular weight based on the ladder was found to be 25 kDa which is similar to the concentration found from the purification lab showing the labs did work in some sense. <span style="font-family: Arial,sans-serif; font-size: 10pt; line-height: 1.5;">All the steps learned from this lab are very important later on when more detailed lab will be performed and protein will need to be characterized again. Then by GOLD ligands can be found that bind to the protein to make a drug inhibitor.

__ References: __
 * 1) <span style="font-family: Arial,sans-serif; font-size: 10pt;">Protein production and purification. Nat Methods, **2008**, 5(2):135-46.
 * 2) <span style="font-family: Arial,sans-serif; font-size: 10pt;">Gjestorff, M. F., Besir, H., Larsen, M. R., Ditzel, H. J., Expression, purification and characterization of the cancer-germile antigen GAGE 121: A candidate for cancer immunotherapy. //Elsevier//, **2010**, Vol. 73, (2), 217-222.
 * 3) <span style="font-family: Arial,sans-serif; font-size: 10pt;">Protein Expression and Purification. European Molecular Biology Laboratory, **2013.** []