JULIA+C

__ Title: __ “Mom, why am I purple?” Expression, purification, and characterization of pGEM-gbr22 plasmid in BL21(DE3) E. Coli cells

I__ntroduction__:

 Proteins have a wide range of applications in medicine. They often serve as drug targets in infectious diseases and can also be administered therapeutically as in the case of insulin [1]. In both cases, large amounts of it must be made available for use. Enormous quantities of insulin are used by diabetes patients to regulate their blood sugar, and pharmaceutical companies require large amounts of specific proteins to use as targets in in-vitro assays of libraries of chemical ligands. These demands are met by using E. Coli bacteria to produce large quantities of a protein by inserting a plasmid that codes for the overexpression of a target protein into a colony of bacterial cells and then extracting the protein from it through purification [2]. This process is relatively cheap and cost effective. Large amounts of protein-expressing bacteria can be grown from a single colony.

 The objective of this lab was to make a large amount of a purple protein through bacterial overexpression, separate and purify the protein from the bacteria using an Ni/NTA resin/buffer, and determine the purity of the final protein sample.

__ Materials & Methods: __

Expression:  Bacterial E. Coli BL21 cells were transformed with pGEM-gbr22 through heat shock and grown on an ampicillin-infused agar plate. The bacterial solution was then allowed to grow in a shaking incubator and turned purple once it started expressing the plasmid (Sample 1). (if you are referencing an image in the paper, make sure to use "Figure: .." i.e don't assume we know what you are referencing)

Purification:  To isolate the protein from the bacterial solution, the solution was centrifuged in an Allegra X-15 at 4 degrees C, 5000 rpm for 10 minutes. This separated the bacteria and protein from the LB and ampicillin and left a bacterial/protein pellet that was treated with lysozyme to break down bacterial cell walls. PBS solution and cyanase was then added to the solution, which was centrifuged at 4 degrees C for 20 minutes at 14000 rpm in a Biofuge Pico Heraeus centrifuge. The top supernatant layer (Sample 2) full of soluble proteins was extracted and purified through a syringe filter then added into a chromatography tube with Ni-NTA resin/buffer (Sample 3). The solution was further purified with wash buffer (1xPBS and20mM imidazole) (Sample 4). Afterwards, the protein was released from the nickel by adding elution buffer (1xPBS and 250mM imidazole). This was done twice, and both samples of both solutions that passed through were saved (Sample 5 and 6). These samples (Elution 1 and 2) were then analyzed with an ND1000 nanodrop at 280 and 574 nm.

Characterization:  Samples 1-6 collected throughout the experiment were then put into an SDS-PAGE electrophoresis module for 25 minutes at 200 V. The gel produced was washed in an orbital shaker 3 times for 5 minutes, left overnight in the shaker, and then dried under Whatman filter paper.

__ Results: __

Figure 1: Experimental Plate – LB + Amp + //E. coli// BL21 with pGEM-gbr22 plasmid. Approx. 150 colonies Figure 2: Control Plate – LB + Amp + //E. coli// BL21 Figure 3 (top): Post-shaking incubator bacterial sample – LB + Amp + E. Coli with pGEM-gbr22 plasmid Figure 4 (bottom) : Post-centrifuge wet protein and bacterial sample –E. Coli with pGEM-gbr22 plasmid

Figure 5: Elution 1 and 2 – purple protein with 1xPBS and 250mM imidazole Figure 6: Trial 1 Nanodrop of Elusion 1 at 280 nm: 0.457. Graph of wavelength vs Absorbance. Figure 7: Trial 2 Nanodrop of Elusion 1 at 280 nm: 0.412. Graph of wavelength vs Absorbance.

The Beer’s Law equation can be rearranged from A = ξbc to c = A/(ξb). b is a constant 1 cm, the ξ is given (38850), and the absorbance is averaged from the two trials (0.444), giving c = 0.444/(38850*1) = 1.143x10-5 mol/L. (1.143x10-5 mol/L)*(25794.2 grams of protein/mol) = 0.295 mg/mL (0.295 mg/mL)*(5 mL) = **0.763 mg of protein**

Figure 8: SDS-PAGE Gel Electrophoresis of His-tag purified gbr-22 protein. Ladder: Thermo Scientific Prestained Protein Ladder 10-170kDa. Samples 1-6. Figure 9: Thermo Scientific Prestained Protein Ladder 10-170kDa

__ Discussion: __

Lysozyme was used in this experiment to break down bacterial cell walls as the first step in extracting the purple protein from the bacteria. Cyanase was used to break down the DNA/RNA in the bacteria. Ni/NTA resin buffer is part of a HIS tag system that was used in the protein purification step of this lab. The pGEM-gbr22 plasmid also coded for a His tag on the purple protein. The Ni/NTA resin buffer consisted of a Nickel attached to a large bead that did not pass through the chromatography tube filter. When the two solutions were mixed, the nickel attached to the protein’s His tag, kepping the target protein from filtering through until all of the other bacterial proteins had done so. Sample 1 contained a solution of E.Coli cells overexpressing the purple protein. Sample 2 contained the supernatant (top liquid layer) of the centrifuged lysed cells that contained all soluble proteins. Sample 3 contained the flow-through from the chromatography tube after the supernatant was flushed with Ni/NTA resin/buffer mix. Sample 4 contained the flow through from the chromatography tube flushed with 20mM imidazole wash buffer. Sample 5 contained Elution 1, 250 mM Imidazole elution buffer and the unanchored target protein. Sample 6 contained Elution 2, elution buffer and the remnants of purple protein not washed out by Elution 1. The main difference between the wash and elution buffers was that the wash buffer had a smaller concentration of imidazole and was used to filter through excess protein while the more highly concentrated elution buffer’s purpose was to detach the protein from the Ni/NTA resin/buffer. During the expression step of the experiment, the growth of the bacteria on the ampicillin-infused plate indicated that it had absorbed the plasmid, which also contained the gene for ampicillin resistance. The bacterial solution did turn purple and the final weight of the bacterial pellet produced after centrifugation was 0.55 grams. During the purification step of the experiment, it was found that Elusion 1 had 1.475 mg of protein at 280 nm and 0.763 mg of protein at 574 nm. The calculated amount of protein decreased two-fold because 574 nm is such a large wavelength that only several very specific proteins are detected in this spectrum. During the characterization step of the experiment, it was found that the MW of the protein was approximately 30 kdaltons. The Elution 1 and 2 lines on the gel run were very clear except for a thick line at 30 kdaltons indicating the target purple protein and a very vague line around 13 kdaltons. The gels showed that the sample consisted mostly of our sample with trace amounts of another protein. Numerically, it was approximately a 95% purity. There were many chances for error in this experiment. The l. ysozyme and cyanase could have not lysed all the bacterial cells, thereby decreasing the total amount of protein extracted. Some bacterial debris may have been taken up with the supernatant after centrifugation, thereby skewing the nanodrop results. During purification, the Ni/NTA resin/buffer may have also tagged onto other proteins and contaminated the elution samples. Also, the electrophoresis run may have been faulty and have marked the molecular weight of the purple proteins incorrectly, skewing the results.

__ Conclusions: __

 In this lab, the purple gene pGEM-gbr22 was inserted into E. Coli as a plasmid and overexpressed. It was then purified by several steps of filtration using Ni-NTA resin/buffer. A gel was then made with samples of 6 steps in the procedure to determine the purity of the final elutions. In the gel run, samples 5 and 6 had a very high purity, indicating that the spectrophotometry data was very good estimate of the amount of protein (0.763 mg at 574 nm) in the final solution. The gel run also helped estimate the MW of the protein, which turn out to be about 30 kdaltons.  The techniques used in this lab will all be applied in the VDS stream to purify and determine the purity of a sample of a target protein made through bacterial overexpression. This technique is a cheap and efficient way of making large quantities of a protein.

__ References: __


 * 1) Sahdev, S.; Khattar, S. K.; Saini, K. S., Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies. //Mol Cell Biochem// **2008,** //307// (1-2), 249-64.
 * 2) Dong, L.; Zhang, X.; Yu, C.; Ren, J.; Hou, L.; Fu, L.; Yi, S.; Chen, W., Expression and purification of recombinant proteins based on human prostate stem cell antigen and heat shock protein-70. //Exp Ther Med// **2013,** //5// (4), 1161-1164.