FENG+G

__Title__: Expression, Purification, and Characterization of pGEM-gbr22 using E. coli, affinity chromatography, and gel electrophoresis.

__Introduction: __

Many times in drug screening, the target protein is too small in concentration. Therefore, a more efficient method is required to isolate target proteins in great quantities so that when studying the effects of drugs on the activity of proteins, only the target protein, which in this study is gbr-22, is studied. A three step method is utilized to accomplish such task. The protein must first be over expressed by inserting a plasmid call pGEM-gbr22 that encodes the gene for the protein into E. coli. By over expressing the protein of interest in bacteria, proteins that are low abundance in their native organism can easily be purified for crystallography and enzyme inhibition assays. This technique also allows one to study human proteins without having to use human tissues. The second step is purification of the over expressed proteins using affinity chromatography. Affinity chromatography separates proteins on the basis of a reversible interaction between a protein and a specific ligand, Ni-NTA, coupled to a chromatography matrix [1]. With a high selectivity and a high capacity for the proteins of interest, high recovery levels of the target protein can be retrieved [1]. To elute the target molecule from the affinity medium the interaction can be reversed using a competitive ligand, imidazole [2]. Target molecules can be purified from complex biological mixtures and small amounts of biological material can be purified from high levels of contaminating substances. A gel electrophoresis is then conducted to validate the purification process. (Great overview!)

The objective of this lab was to only harvest the over expressed protein gbr-22 from E. coli BL21 (DE3). The hypothesis is that if the protein expression and purification process is conducted properly, then only one protein, gbr-22, should be seen in the final elution sample after gel electrophoresis.

__Materials & Methods: __ 1. Protein expression 25 µl of competent bacteria cells were first heat shocked with 2 µl plasmid pGEM-gbr22 at 42 degrees Celsius for 45 seconds so that the plasmid encoding gbr-22 and Ampicillin resistance can enter the bacteria. 200 µl SOC media was then added to the cell mixture. After 30 minutes in the 37 degrees Celsius incubator, 50 µl of bacteria mixture were dispensed on Ampicillin positive agar plates and stored in 37 degrees Celsius incubator over night. The starter culture containing pGEM-gbr22 was then transferred into Erlenmeyer flask containing LB and Ampicillin. The flask was then shook in a shaking incubator for 24 hours. Ampicillin resistance was given to bacteria who (do not personify bacteria)  also received gbr-22 gene to ensure that the only bacteria growing on the Ampicillin Agar plate was the ones who received the plasmid. A control group was also made where competent bacteria cells with no plasmid were grown on another Agar plate containing Ampicillin. This is to validate that the bacteria who did not receive the plasmid would actually not be able to grow on an Ampicillin plate. After the control is validated, the transformed bacteria cells were then grown in a large culture to then be harvested (500µl taken as sample 1).

2. Protein Purification The bacterial cells were first lysed open, and Cyanase was then added to digest the DNA. After centrifugation, the supernatant (50 µl taken for sample 2) was then isolated into a 15ml conical tube. 10x PBS and 1M imidazol and nanopure water were dispensed into the 15ml conical tubes. The lysate was then syringe filtered through a 0.45 µm PES syringe filter. To bind the protein to the resin, 0.25 ml of Ni-NTA resin and 0.25 ml of buffer were mixed into the column. After the material has settled in the column, the solution was drained into the “waste” tube (50 µl taken for sample 3). The Ni-NTA resin were then washed with 5 ml of 20 mM imidazole in 1x PBS to remove the proteins that were loosely bound to the resin (50 µl taken for sample 4). Finally, the gbr22 protein was eluted from Ni-NTA resin by flushing the column with 5 ml 250 mM imidazole(50 µl collected first time is sample 5, 50 µl collected second time is sample 6). The Ni-NTA were stripped (What exactly is being stripped? the Ni-NTA or proteins bound to it?)  with 10 cv water, 10 cv 0.5 M NaOH, and 10 cv water. Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) was used to measure the concentration of the purified protein at 280 nm. (and 574nm)

3. Protein Characterization <span style="font-family: Arial,Helvetica,sans-serif;">A gel electrophoresis was conducted using a Mini-PROTEAN electrophoresis tank and lid (Bio-Rad, Hercules, CA). The 6 samples were placed in heat block for 90 degrees Celsius for 5 minutes, after which the tubs were then centrifuged for 2 minutes at 5,000 rpm. The Mini-PROTEAN electrophoresis tank was set up correctly according to the equipment set-up protocol. Once the samples and the MW standard were loaded into the wells, the gel was set to run for 25 minutes at 200 V. The gel (What kind of gel?) <span style="font-family: Arial,Helvetica,sans-serif;"> was then stained using Imperial protein stain for 1.5 hours on an orbital shaker. The gel was then destained overnight using Nanopure water and a small folded tissue. The gel was then dried on Whatman paper covered with Saran Wrap at 75 degrees Celsius for 1.5 hours. (Too long, try condensing)

__<span style="font-family: Arial,Helvetica,sans-serif;">Results: __ <span style="font-family: Arial,Helvetica,sans-serif;">

<span style="font-family: Arial,Helvetica,sans-serif;"> <span style="font-family: Arial,Helvetica,sans-serif;"> <span style="font-family: Arial,Helvetica,sans-serif; line-height: 0px; overflow: hidden;">

<span style="font-family: Arial,Helvetica,sans-serif;">

<span style="font-family: Arial,Helvetica,sans-serif;">Definitely did not have the most bacteria. <span style="font-family: Arial,Helvetica,sans-serif;">

<span style="font-family: Arial,Helvetica,sans-serif;"> <span style="font-family: Arial,Helvetica,sans-serif;"> <span style="font-family: Arial,Helvetica,sans-serif;">A = Ebc 0.037 x 10 = 118.300 x c (show more work and units) c = 0.82 mg/ml

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__<span style="font-family: Arial,Helvetica,sans-serif;">Discussion: __ <span style="font-family: Arial,Helvetica,sans-serif;">Overall, the protein expression, purification, and characterization worked successfully. It can be seen from figure 1 that the starter colonies with Ampicillin resistance and pGEM-gbr22 grew successfully. This was validated by comparing the DNA plate to the non-DNA plate in figure 2, where no starter colonies could be seen even though both Agar plates were incubated over night at 37 degrees Celsius. Figure 3 represents a swab of the computer keyboard, was grown on a regular Agar plate, containing only a few colonies after 24 hours of incubation. The purple color of the large starter culture after 24 hours of incubation demonstrates the successful transformation of pGEM-gbr22 in figure 4. The isolated bacteria cells containing gbr22 can be seen in figure 5 as a pellet after centrifuging. The rich purple color confirms that the gbr22 is successfully over expressed. In figure 6, Elution 1 can be seen in with a light purplish color, representing the gbr22 in elution buffer. It can be noted that Elution 2 in figure 7 is significantly lighter in color than Elution 1. This is because Elution 1 contained the first elution of gbr22 from the Ni-NTA matrix, which means that most of the gbr22 was released into Elution 1. As a result, Elution 2 contained less gbr22 due to the fewer gbr22 that was still bound to the matrix. In figure 8, a Nanodrop spectroscopy was conducted to estimate the concentration of gbr22. <span style="font-family: Arial,Helvetica,sans-serif;">The success of protein expression was strongly due to the use of lysozyme and Cyanase, which broke down the bacterial cell wall and the RNA/DNA so that they could be small enough to be filtered through. The HIS tag system works by utilizing the 6x HIS tag on gbr22 to bind to the Ni-NTA in the column so that when flushed with washing buffers, the target protein will not be filtered through. Once all other proteins have been filtered thorough, a high concentration elution buffer was used to release gbr22 from the Ni-NTA matrix. <span style="font-family: Arial,Helvetica,sans-serif;">The results can be seen from gel electrophoresis in figures 9 and 10, where there is a significant decrease in number of bands from sample 1 to sample 6. This further validated the success of the purification process. However, elution 1 was only about 40% purity because there are two other bands in the same lane as elution 1. In lane 1 is sample 1, which was taken prior to any purification technique. This yielded many bands with various weights of proteins. Lane 2 contains sample 2, which was the soluble fraction taken after the heavier cell debris were centrifuged to the bottom. This however did not reduce the number of heavier protein bands in sample 2. Sample 3 was the flow through when Ni-NTA and the soluble fraction flowed through the column. Sample 4 was the wash with a low concentration of imidazole, which removed the proteins that were loosely bound to the Ni-NTA affinity matrix. A higher concentration imidazole elution buffer was then used to release gbr22 from the Ni-NTA matrix. Sample 5 was taken after running the column with a higher concentration of imidazle, dislodging most of gbr22 from Ni-NTA matrix. This yielded a dark band at 25kDa according to the pre-stained protein ladder standard. Sample 6 was the second flow through of the high concentration imidazole, which yielded a lighter band at 25 kDa. It can be seen that gbr22 at 25 kDa from sample 1 through sample 4 as gbr22 is kept through the filtering process. The size of the protein from the gel is estimated to be around 25 kDa where as it was estimated to be 25 kDa from protein purification. The hypothesis was confirmed in that gbr22 protein was purified at the end and validated through gel electrophoresis. <span style="font-family: Arial,Helvetica,sans-serif;">Sources of Error: <span style="font-family: Arial,Helvetica,sans-serif;">Because of 2 additional bands appeared in sample 5 and 6 after gel electrophoresis, there could be contamination in elution 1 and 2 during the entire lab process. Another source of error was that while loading the samples into the wells, one of the wells was accidentally punctured by a micro-pipette tip, so samples could have leaked between the lanes.( <span style="color: #ff0000; font-family: Arial,Helvetica,sans-serif;">(good point. your discussion has a lot of information. The description of what is in each well should be confined to the caption only. You can talk about the elution samples though)

__<span style="font-family: Arial,Helvetica,sans-serif;">Conclusions: __ <span style="font-family: Arial,Helvetica,sans-serif;"> After successfully the over expressing gbr22 in competent E. coli cells, gbr22 was purified through affinity chromatography technique using Nickel as the ligand for the HIS tag. The successful purification of gbr22 was later verified through gel electrophoresis. The next step towards this project is to determine the source of the contamination in elution 1 and 2 and improve the purification technique for future wet lab protein target screenings. <span style="color: #ff0000; font-family: Arial,Helvetica,sans-serif;">(include something about expression of other proteins for use in enzyme assays.)

__<span style="font-family: Arial,Helvetica,sans-serif;">References: __ <span style="font-family: Arial,Helvetica,sans-serif;">1. Affinity Chromatography: Principles and Methods; GE Lifesciences: Little Chalfont, 2013. <span style="font-family: Arial,Helvetica,sans-serif;">2. Recombinant Protein Purification Handbook: Principles an Methods; GE Lifesciences: Little Chalfont, 2013.