RAMIRO+R

Intro should be more background info on theory or on the technique than restating methods Reduce M&M to most critical details Use past tense, 3rd person (hypothesis) Improve calculations __ Title: __


 * The Triple Threat Protein Lab**

__ Introduction: __

The process of expressing, purifying, and characterizing a recombinant protein is a very useful and stressful activity. Recombinant proteins are used throughout biology for the creation of many proteins and drugs. Their production was once the domain of experts, but the development of simple, commercially available systems has made the technology more widespread. [1] Protein production and purification is a very difficult process because no two proteins are alike. Therefore there is no set parameter to go about producing and purifying all proteins in general. Over the past decade, laboratories have collectively targeted and purified tens of thousands of different proteins from the Eubacteria and Archaea, and thousands from the Eukarya, including fungal, nematode, parasite, plant and human proteins. [1] Every protein has its own unique characteristics and needs its own production and purifying process. The three steps for the protein labs are expression, purification and characterization. In protein expression a recombinant protein is overexpressed in bacteria. This is done by transforming bacterial cells (BL21(De3)) with a DNA plasmid (pGEM-gbr22), growing a starter culture of bacteria overnight to manifest a larger culture, harvesting the cells, and preparing them for purification. Protein purification is cleansing the bacterial protein. This is done by, breaking open the bacterial cells, collecting the soluble proteins, removing the insoluble cell debris by centrifugation, and purifying the protein using an affinity tag and Ni-NTA resin. Several samples are made in this lab that will be used in protein characterization. In protein characterization gel electrophoresis is used to analyze the protein samples from steps 1 and 2. Electrophoresis and spectroscopy are used to estimate the weight and concentration of the gbr22 protein. The overall outcome of these three steps is to produce the final purified protein. I believe that the protein will not be 100% purified at the end of all three steps because there is always human error and miscalculations in every lab.

__ Materials & Methods: __

On day one of protein expression an experimental plate, a control plate and a fun plate will be made. Label all of the plates accordingly. Add 25ul of bacterial cells to 2 transformation tubes and centrifuge them. Now add 1-2ul of plasmid in the experimental tube only. After 30 minutes heat shock tubes in 42oC water for 45 seconds. Wait two minutes and add SOC media and then leave the tubes in the incubator for 30 minutes. Pipette 50ul into each plate and use colirollers to spread the bacteria. Find something disgusting to put on the fun plate and watch it grow. On day two morning the starter culture will be grown in LB supplemented with 100ug/ml ampicillin. Add ampicillin to two tubes of 5ml LB and then add bacteria from each plate into one tube. Place the tube in the incubator and your plates in the 4oC fridge. On day 2 evening the large overnight culture will be made by tranfering 25ml of LB, some ampicillin, and .625ml of the starter culture to a 125ml Erlenmeyer flask. Place the flask in the incubator for 16-24 hours. On day three, if your culture is purple (if it is yellow come back for day four) take a 500ul sample, dispense it into an Eppendorf tube and label it sample 1. Pour the remaining bacteria sample into a 50ml conical tube, label it and make another tube of equal wait in water for a counter balance. Cap the tubes and centrifuge them for 10 minutes at 5000 rpm at 4oC. Afterward save the pellets and discard the liquid. Record the weight of your pellet. In a 15ml conical make a 1x PBS working solution, vortex it, add lysosome and store in the -20oC fridge for purification. To begin protein purification, thaw your tube and then add Benzonase to the tube. Now clarify the lysate (sample 2) and isolate the soluble fraction. Prepare the wash and elution buffers (samples 3 and 4) accordingly and then syringe filter the lysate into a 14ml transformation tube. Next perform the Ni-NTA affinity purification and add the wash and elution buffers. From this elutions 1 and 2 (samples 5 and 6) will be created and stored in the 4oC fridge. Next strip the Ni-NTA by washing it with 30% ethanol. Now use the Nanodrop spectrophotometer to find the concentration of the protein at 574nm. For protein characterization, first prepare sample 1 and then centrifuge it. Then prepare samples 2-6 and place them all in the gel cassettes for electrophoresis. After electrophoresis stain the protein gel and leave it overnight on the shaker. The next day, dry the gel and then take the gel out and analyze the results.

__ Results: __



Figure 1. The bacterial starter culture (LB + AMP, BL21 (DE3), pGEM-gbr22) on Day 2 morning of Protein Expression.

Figure 2. The Erlenmeyer flask with bacteria (LB + AMP, BL21 (DE3), pGEM-gbr22) from day 3 of Protein Expression.

Figure 3. The 50ml conical tube from Protein Expression. The purple pellet is bacteria (LB + AMP, BL21 (DE3), pGEM-gbr22) that were centrifuged. The weight is .61g of wet pellet.

Figure 4. Elutions 1 and 2 in 15ml conical tubes from Protein Purification. Elution 1 is the light purple and elution 2 is clear.

Figure 5. Nanodrop spectrophotometer reading of elution 1, trial 1. Absorbance vs. Wavelength. Protein 280nm settings.

Figure 6. Gel before drying from Protein Characterization. The ladder is on the left in lane 1, lane 2 is the soluble fraction, lane 3 is the flow through, lane 4 is the wash, lane 5is elution 1, lane 6 is elution 2, and lanes 7-9 are probably accidental mixing of lanes that could be a source of error.

Figure 7. Dry gel from Protein Characterization. The ladder is on the left in lane 1 (from bottom to top), lane 2 is the soluble fraction, lane 3 is the flow through, lane 4 is the wash, lane 5 is elution 1, lane 6 is elution 2, and lanes 7-9 are probably accidental mixing of lanes that could be a source of error.

Figure 8. Bio-Rad molecular weight standards for proteins.

Beer's Law:

A=Ebc

__ Discussion: __

Lysozyme is an enzyme that was added either at the end of protein expression or at the beginning of protein purification to digest the cell wall of the E.coli. In purification benzonase/cyanase was because it digests DNA/RNA of E. coli. Overall the reason both were added was to separate bacterial protein from all other substances. The HIS tag works binding to cations like nickel that can be restrained on a column such as Ni-NTA resin. The protein is then released from the Ni-NTA resin with the addition of imidazole, by competing with the histidine for binding. There were 6 samples collected from the 3 labs that are seen on the dry gel. Sample 1 is from protein expression and contains E. coli cells, proteins, and cell debris. Sample 2 is from purification and only contains the soluble fraction with the lysozyme. Sample 3 is Ni-NTA resin flow through from purification. This is after the syringe filtering and contains proteins that did not stick to the nickel. Sample 4 is the wash and contained proteins that were loosely attached to the Ni-NTA resin. Sample 5 is the elution 1 buffer and sample 6 is after the elution 1 buffer. There are lighter bands were found in wash than in elution one. The lighter bans in elution 1 show that the protein is not 100% pure. I would estimate the purity of the protein to be around 35%. When comparing the bands to the Molecular Weight Standards it is approximately to approximately 30 kDa. Throughout all three labs there are many possible sources for error that include, incorrect concentrations, calculations, and techniques. For example the incorrect amount of PBS and imidazole could have been added during protein expression.

__ Conclusion: __

Bacterial cells (BL21(De3)) was combined with a DNA plasmid (pGEM-gbr22) to form recombinant DNA. The recombinant DNA was then grown in a starter culture of bacteria overnight to manifest a larger culture, harvested and prepared for purification. The protein was purified breaking open the bacterial cells, collecting the soluble proteins, removing the insoluble cell debris by centrifugation, and purifying the protein using an affinity tag and Ni-NTA resin. Several samples are made in this lab that will be used in protein characterization. In protein characterization gel electrophoresis is used to analyze the protein samples from steps 1 and 2. Electrophoresis and spectroscopy are used to estimate the weight and concentration of the gbr22 protein. I believe that the protein will not be 100% purified at the end of all three steps because there is always human error and miscalculations in every lab.

__ References: __

1. Acton, T.; Albeck, S.; Almo, S.; Anderson, S.; Arrowsmith, C.; Atwell, S., Protein production and purification. //Nature publishing group.// **2008**. (2), 135-46.

2. Ponko, S.; Bienvenue, D.; Protein Tracker: an application for managing protein production and purification. //BMC Research Notes//. **2012**, (5), 224.

3 European Molecular Biology Laboratory website []