SHANNON+R


 * __ Title: __** Protein expression, purification, and characterization of pGEM-gbr22.

Recombinant proteins are vital components of research in biomedical and biological sciences. However, since every protein is different, the methods vary for protein production and purification [1]. In this case, the protein pGEM-gbr22 was expressed using BL21(DE3), a strain of //E.coli// bacteria. This is due to the fact that //E.coli// is typically a very good system for the production of many proteins [2]. Next, the protein should be purified in a well-buffered solution, and, lastly, the appropriate affinity tag should be used to isolate it if needed [1]. The objective of this lab was to effectively express and purify the recombinant protein pGEM-gbr22 so that it could be characterized. The protein is hypothesized to be characterized well, and for pGEM-gbr22 to be successfully isolated from the cell.
 * __ Introduction: __**

__Expression__ – First, 25 µL of competent bacterial cells were added to 2 transformation tubes, while 1 µL of plasmid DNA was added to only one, and then left on ice for 30 minutes. Next, the tubes were heat shocked in a 42°C water bath for 45 seconds and cooled for 2 minutes. 200 µL of SOC media was added to the tubes, and they were shaken in the incubator for 30 minutes at 37°C at about 250 rpm. 50 µL of bacteria/SOC mixture from the tube was placed onto each plate (one control, one DNA), spread evenly, and incubated overnight. 10 µL of ampicillin was added to 5 mL of LB in a sterile culture tube. A single colony from the DNA plate was added to the tube, and shaken in an incubator at 37°C at 200-350 rpm for about 8 hours. Later, 25 mL of fresh LB, 50 µl of ampicillin, and 0.625 of a starter culture was added to a sterile 125 mL Erlenmeyer flask, and incubated overnight. A sample of the purple media from the flask was collected and the rest was transferred into a 50 mL conical tube. It was centrifuged for 10 minutes at 5,000 rpm at 4°C. Afterwards, 2.5 mL of 1x PBS solution was added to the tube containing just the pellet, and vortexed. 50 µL of lysozyme was then added to the tube, vortexed again. The tube was placed into the -20°C fridge until the next procedure. __Purification__ – The 50 mL conical tube was thawed in a beaker of water, and 2 µL of Cyanase was added to it. After being inverted then incubated at room temperature for 15 minutes, it was centrifuged for 20 minutes at 14,000 rpm at 4°C. Next, a sample of the supernatant was taken, and the rest was transferred to a 15 mL conical tube leaving the pellet behind. The lysate was then syringe-filtered. Next, 0.5 mL of Ni-NTA resin/buffer mix was added to the tube, and incubated at room temperature for 15 minutes, while mixing occasionally. A 20 mL Bio-Rad chromatography Econo column was used to isolate the protein by removing the cap and allowing the contents from the tube to flow through, as well as a wash buffer and elution buffer, keeping the level of PBS buffer above the resin. After each flow through a sample was collected and stored. A Nanodrop spectrophotometer was then used to examine the Elution 1 and 2 samples with the absorbance at 280 nanonmeters, then 574 nanometers. __Characterization__ – This involves the samples taken from the previous steps. The first sample was centrifuged for 5 minutes at 5,000 rpm. Afterwards, the pellet alone was re-suspended in 200 µL of water and 40 µL of loading buffer. About 8 µL of the loading buffer was added to the rest of the samples. All of the samples were heat blocked at 95°C for 5 minutes, then centrifuged for 2 minutes at 5000 rpm. The electrophoresis module was assembled and 20 µL of each of the samples were loaded into the wells starting with the second well, after they were cleared using a 20 gauge needle. The gel ran for 25 minutes at a voltage of 200. Afterwards, the gel was placed into a dish with nanopure water and washed 3 times for 5 minutes. After the water was drained, about 30 mL of protein stain was added, and the gel was washed for about 1 hour. The gel was then rinsed twice and washed overnight. The next day, the gel was dried on a drying bed at 75°C on gradient cycle for 1.5 hours.
 * __ Materials & Methods: __**

__ Results: __





yield= concentration x volume in the tube concentration=absorbance/(extinction coefficient x path length)

Yield of Elution 1 (Trial 1) at 280 nm: 0.0000265 mg Yield of Elution 1 (Trial 1) at 574 nm: 0.00000871 mg Yield of Elution 2 (Trial 1) at 280 nm: 0.0000231 mg Yield of Elution 2 (Trial 1) at 574 nm: 0.00000972 mg

The yield results between Elution 1 and 2 are very similar to each other. On the other hand, the results from the gel were affected by errors in the experiment. For instance, the gel was over-stained, which contributes to the fact that the gel is purple in color while it should be clearer in color. Also, the gel was ripped while clearing it with the syringe. Therefore, in the lanes for the second samples of 5 and 6 have more than one protein strongly expressed. In this case, only the first sample 5 was counted toward determining the purity of the protein which was about 35%. The reason for the addition of lysozyme was to break down the cell membrane surrounding the protein. Cyanase was used to digest the DNA/RNA in the cell. Next, the HIS tag system allowed the protein to be isolated; the histidine on the protein bound to the nickel, however the imidazole overpowered this binding due to its histidine wanting to be bound to the nickel also. Therefore, when the flow through occurred, the protein wound up inside the waste. The wash buffer contained less imidazole than the elution buffer, so the protein escaped more readily when the elution was rather than when the wash was. Sample 1 contained the bacteria before a cell pellet was made. Sample 2 held the cell after the breaking of the membrane and the DNA/RNA was digested by the Cyanase. In sample 3 was the waste from the flow through in the column before any buffers were added; excess proteins that also existed in the cell. Sample 4 was the waste collected after the wash buffer was added to the column; the protein pGEM-gbr22 should not be in this sample. Sample 5 contained the pGEM-gbr22 protein; this happened after elution 1. Sample 6 contained very little of the protein, if any; this occurred after elution 2. The size of the protein according to the protein ladder referenced was about 15 kDa and the size of the protein according to the protein purification was about 0.0000265 mg. These differ by about 10 orders of magnitude. This could be due to errors during the gel electrophoresis.
 * __ Discussion: __**

The protein pGEM-gbr22 was expressed, purified, and characterized. The protein was successfully eluted; however, the gel could be redone to produce better results. In the future, the experiment could be repeated with the procedures being more successive. In addition, the protein could then be used as a target for drug inhibition.
 * __ Conclusions: __**


 * __ References: __**
 * 1) Gräslund, S.; Protein production and purification//. Nat Methods// **2008,** 5, (2), 135-46.
 * 2) Kusnadi, A.R., Hood, E.E., Witcher, D.R., Howard, J.A., Nikolov, Z.L.; Production and purification of two recombinant proteins from transgenic corn. //Biotechnology Progress// **1998,** 14, 149-155.