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= Characterizing the Purification of Expression: Lab Report for Protein Labs = toc

Introduction
In order to propel biological research there are methods that isolate the molecules involved. As such, for research in to proteins and their inhibitors, researchers must produce recombinant proteins that are pure of other molecules. This is achieved through protein expression, purification and characterization. Protein Expression is the process of introducing the DNA of the specified protein as a plasmid, into a bacteria culture. This host bacterium allows for rapid growth of the protein in question, over expressing the desired protein. This is essentially transforming the Bacteria giving it properties it otherwise wouldn’t have to suit the needs of the researcher. By growing the bacteria with ampicillin resistance, this assures a pure culture devoid of bacteria undesirable otherwise. The next step is purification, which is the process of taking these bacterial cells and breaking them down in order to obtain the protein itself. Testing with the bacteria and other molecules is not beneficial to the researcher; as such this is a very crucial step. By using lysosomes to break down the bacteria and filter it through various means, a pure protein substance can be achieved. Characterization is the process that allows for indication of how pure a protein sample is. Using gel electrophoresis, the researcher can use previous sample of the protein before purification, and the pure protein sample, the purity of the sample can be gauged by the limited amount of other small molecules that appear in the electrophoresis gel. The more pure a substance is, the better it can be used in further experiments. In these sets of labs, the process of isolating a protein is explored using the protein meffCP-gbr22, a fluorescent protein obtained from a coral species // Monitpora efforescens // from the Great Barrier Reef, and over expressing it in // E Coli //bacteria. This will proceed into the purification and characterization of the protein as a means of demonstrating the process.

Protein Expression
· Ice bucket · 2 x 14 mL clear, sterile round-bottom tubes · 37 degrees Celsius incubator · Gas burner · 42 degree Celsius water bath · Pipette · Pipette tips · Colirollers · 2 LB Agar plate w/o antibiotic · Competent cells on ice · Plasmid DNA on ice · LB media · SOC media

Protein Purification
· Ice bucket · Beaker of room temp. water · 1 M Imidazole · 10x PBS · 1.7 mL centrifugal tubes · 2 (two) 10mL round-bottom tubes · 4 (four) 15mL conical tubes · Bio-Rad Econo chromatography column · Ring stand · Clamps · Ni-NTA resin · Benzonase (keep iced)

Protein Characterization
· Mini-PROTEAN electrophoresis tank and lid · Power supply and leads · TGS running buffer · Bio-Rad precast polyacrylamide gel · 100µL 6x gel (or sample) loading gel · Protein samples #1-6 from Expression and Purification labs · Molecular Weight standards · Plastic container w/ lid · Imperial protein stain · Whatman paper · Cellophane

Methods
The Protein Labs were done on a bi-weekly basis starting with the expression of the recombinant protein. This was done over a three day period which began with transforming the competent bacterial cells with the meffCP-gbr 22 protein plasmid. Two plates were made, a control with ampicillin within the agar and the bacterial cells without a plasmid, and the experimental plate with the same plate, except exposed to the fluorescent plasmid. This assures that were growing only the bacteria with the fluorescent plasmid.The 50 ul bacterial cells were put in to transformation tubes, one with added plasmid DNA. These tubes were placed on ice then heat shocked and placed on the agar plates. On the second day, the grown amp resistant bacteria were transferred from the plate to two flasks and grown overnight. The third day, a sample was taken and stored and the flask grown bacteria were transferred to conical tubes, centrifuged for 10 minutes at 5,000rpm at 40C down to just pellets. The pellets were transferred to conical tubes and re-suspended in a solution with lysozyme stock to be used for the next lab. The next lab was the purification of the protein which began with lysing the bacterial cells. In order to make the resulting solution less viscous, Benzonase was added and incubated. The solution was centrifuged for 30 minutes at 10,890 at 40C. The resulting liquid supernatant was filtered through a Bio-Rad chromatography column with a Ni-NTA affinity buffer. After collecting the waste and wash, two Elution solutions were collected and saved as samples. A Nanodrop spectrophotometer was used to test the the concentration of protein within the elution solustions by using Beer's Law. The final lab characterized the protein by comparing samples collected throughout the first two labs. This was achieved through a gel electrophoresis. The electrophoresis module was set up with a Gel cassette and TGS buffer. Loading buffer was added to the samples and placed, along with a molecular weight standard, in to the wells. The electrophoresis was run for 25 minutes at 200V. The gel was then stained and dried.

Results










Calculations for absorbance values:  UV/VIS:  A= εbc @ 280nm  .054 = (38850L/mol x cm) (1cm) (c)  C= 1.38 x 10^-6 mg/ml  A= εbc @ 574nm (maximum wavelength)  .105 = (118300L/mol x cm) (1cm) (c)  C=8.87 x 10^-6 mg/ml





Discussion
The protein is almost completely pure aside from a single faint band so the estimate protein content could range anywhere from 80-90% of the molecular contents being the protein. The weight of the protein was determined to be ~30Kda as it is neither at the 35 nor the 25 mark of the molecular weight standard; however it does shy closer to the 25 mark. The absorbance data from the Nanodrop seemed to be in line with what was to be expected. The calculated yield of protein within the elution solution was determined to be ~1.2mg/ml. It should be noted that samples 1, 3 and the wash were obtained from another lab’s set of samples as the samples in this lab could not be located.

Conclusion
This lab yielded a successful sample of meffCP-gbr22 protein obtained from //Montipora efforescencs// achieved through overexpressing the protein, purifying it, and then identifying thorough characterization. The lab was a successful tool in teaching how protein samples are created and the long processes involved. This protein can be used in future experiments to study how proteins are inhibited. The next step would be an enzyme assay that would look at how inhibitory action is measured and the steps involved in order to inhibit a protein.