Urvashi+M

__Expression, Purification, and Characterization of pGEM-gbr22 __

__ Introduction: __ Protein production and purification is a complex process. First, the gene encoding the protein of interest must be cloned and inserted in an expression plasmid. Then a bacterial strain must be transformed with the plasmid. //E//. //coli,// particularly the strain BL21(DE3), is an effective and commonly used expression host for protein production(1). Proteins may be tagged at the N- or C- terminus with affinity tags such as hexahistidines for more efficient purification. The heat shock method may be used on chemically competent bacterial cells for transformation (2). Selective markers such as ampicillin resistance are often employed to identify successfully transformed colonies. One of these colonies may then be used to grow a starter culture. Induction of protein expression and harvesting of the cell pellet follows (2). The protein of interest can be extracted from the pellet using chromatography methods and then purified with gel filtration. This experiment will involve transformation of //E. coli// BL21(DE3) with a plasmid vector encoding for the purple protein gbr22, expression of the protein in a large culture, and subsequent purification and characterization of the protein. If the procedure is done correctly, then the protein will have a high purity and high concentration.

__ Materials & Methods: __ A transformation tube containing 25 µl of competent //E. coli// BL21(DE3) cells(New England BioLabs) and 2 µl of pGEM-gbr22 was left on ice for 30 minutes, heat shocked in a 42°C water bath for 45 seconds, put on ice for 2 minutes, had 200 µl of SOC media added, and was put in a 37°C shaking water bath for 30 minutes at 250rpm. The bacteria was plated on bactoagar dishes and stored in a 37°C incubator overnight. The next morning, a culture tube containing a transformed bacterial colony, 10 µl of ampicillin and 5 mL of LB was prepared and placed in a shaking incubator at 37°C at 300rpm for 8 hours. A flask containing 25mL LB, 50µL of ampicillin, and 625µL of starter culture was placed in a shaking incubator at 37°C at 300rpm for 20 hours. 500 µL of the culture was saved as Sample 1 and the rest was centrifuged for 10 minutes at 5000rpm at 4°C. The pellet was resuspended in 2.5 mL of 1x PBS and 50 µL of lysozyme, and was stored in a -20°C freezer. The cell suspension was thawed, had 2µL of Benzonase added, and was microcentrifuged for 20 minutes at 14000 rpm at 4°C. 50 µL of the supernatant was saved as Sample 2 and the rest was disposed. The lysate was filtered through a syringe filter and had .5 mL ni-NTA added. The mixture was added to a chromatography column (Bio-Rad), then Wash Buffer, then Elution Buffer twice. 50 µL of flow through after each step was collected as Samples 3-6. The Nanodrop spectrophotometer was used to measure absorbance of the purified protein at 280 nm and 574 nm. Sample 1 was microcentrifuged for 5 minutes at 5000rpm and resuspended in 200 µL of water and 40 µL of loading buffer. Samples 2-6 had 10 µL of 6x loading buffer added and all samples were put on heat block at 95°C for 5 minutes and centrifuged for 2 minutes at 5000rpm. MW standards(Fermentes, SM0671) and Samples were loaded into wells of gel(Bio-Rad) and gel was run for 25 minutes at 200V. The gel was removed, stained with Imperial protein stain, washed overnight, and dried on Whatman filter paper on a drying bed for 75°C for 1.5 hrs.

__ Results: __


 * Figure 1a.** Culture plate containing ampicillin, bacterial organism __BL21 (DE3)__, and plasmid vector __pGEM-gbr22__. The bacterial colonies appear a bright purple color because the inserted plasmid encodes for a fluorescent protein originally cloned from a coral from the Great Barrier Reef. The culture plate demonstrates that transformation of the __E. coli__ cells was successful because the cells are expressing the purple protein. There are several hundred colonies on the plate.

Culture plate containing ampicillin and bacterial organism __BL21 (DE3)__. Since this plate had bacteria without a vector containing resistance to ampicillin, no growth is seen. This plate serves as a control to the plate with the bacteria containing the vector. There are zero colonies on the plate.
 * Figure 1b.**

Culture plate containing bactoagar and bacteria from coughing into the plate. Here, minimal growth is seen. No plasmid vectors were introduced to these bacteria, so they are not colored. This plate served as the 'Fun' plate. There are nine colonies on the plate.
 * Figure 1c.**


 * Figure 2.** Flask containing __E. coli__ bacterial culture that was grown over night in a shaking incubator at 300rpm and 37 degrees Celsius. The culture contains LB, Amp, __BL21 (DE3)__, and __pGEM-gbr22.__ Since the culture was purple, the cells were ready to be harvested.


 * Figure 3.** Purple pellet of __E. coli__ cells obtained after centrifuging the culture (at 5000rpm at 4 degrees Celsius for ten minutes) and decanting the liquid. The wet pellet weight was measured to be .36 grams.


 * Figure 4**. 5 mL each of Elution 1 and Elution 2, obtained from the last two Elution Buffer flows through the Econo chromatography column(BioRad). The elution buffer contained a high concentration of imidazole, which caused the gbr22 protein to be released from the Ni-NTA resin. Elution 1 was purple while Elution 2 was clear.


 * Figure 5.** The two graphs show the absorbance spectra for for gbr22, as collected by the Nanodrop spectrophotometer. The top graph shows Run 1 and the bottom graph shows Run 2. Measured at 280 nm, the absorptions for the runs were .68 and .71, with an average of .695.


 * Figure 6.** The two graphs show the absorbance spectra for for gbr22, as collected by the Nanodrop spectrophotometer. The top graph shows Run 1 and the bottom graph shows Run 2. Measured at 280 nm and the maximum wavelength of 574 nm, the absorptions for the runs were .55 and .54, with an average of .545. The peak in the graph at 574 nm demonstrates maximum absorbance occurs at that wavelength.

A= εbc MW gbr22= 25794.2 g/mol ε gbr22= 38850 m-1cm-1
 * Determining protein yield: **

.695=38850(1) * c c= 4.61 x 10-4 mg/mL 5mL * (4.61 x 10-4)mg/mL= 2.30 x 10-3 mg 545=11830(1) * c c=1.18 x 10-3 mg/mL 5mL * (1.18 x 10-3)mg/mL = 5.9 x 10-3 mg
 * <span style="font-family: Arial,sans-serif; font-size: 10pt;">At 280nm: **
 * <span style="font-family: Arial,sans-serif; font-size: 10pt;">At 574nm: **

Lane 1 contains the MW standard<span style="font-family: Arial,sans-serif; font-size: 10pt;">(Fermentes, SM0671), lanes 2-7 contain Samples 1-6(lab partner's), and lanes 8-10 contain a second set of Samples 4-6(own).
 * Figure 7.** Above, wet gel obtained from gel electrophoresis of Samples 1-6, which contained the purple protein of interest, gbr22. Below, dried gel obtained by placing wet gel on Whatman paper, wrapping it with saran wrap, and drying on a drying bed at 75 degrees Celsius for 1.5 hrs.


 * Figure 8.** Fermentas PageRuler Molecular Weight Standard, SMO671. This ladder was used as a reference for the gel that was run in the experiment and was used to determine protein purity.

<span style="background-color: white; font-family: Arial,sans-serif; font-size: 10pt;">Using the MW standards <span style="font-family: Arial,sans-serif; font-size: 10pt;">(Fermentes, SM0671) as reference, the MW of the purified protein was estimated to be about 25kDa. The presence of four or five other protein bands in the Sample 5 lanes indicated that gbr22 was not very pure, with an estimated purity of about 20%. <span style="font-family: Arial,sans-serif; font-size: 10pt;">Lysozyme, which digests cell walls, and Benzonase, which digests DNA/RNA, were added to the cell suspension in order to lyse the bacterial cells so they could release the desired soluble protein. <span style="font-family: Arial,sans-serif; font-size: 10pt;">Sample 1 contained bacterial starter culture (in log phase). Sample 2 contained soluble fraction of the lysate. Sample 3 contained flow through waste. Sample 4 contained washed protein solution. Sample 5 had eluted protein solution. Sample 6 had protein solution eluted twice. <span style="font-family: Arial,sans-serif; font-size: 10pt;">The elution buffer had a higher concentration of imidazole than the wash buffer, allowing more protein to be released. The his-tag system involves fusing a histidine affinity tag, or number of histidine residues, to a protein. It allows for the protein to be purified with a simple immobilized metal affinity chromatography technique(1). <span style="font-family: Arial,sans-serif; font-size: 10pt;">Sources of error include contamination, miscalculations, inaccurate readings, imperfect solutions, and incorrect measurements.
 * __ Discussion: __**

<span style="font-family: Arial,sans-serif; font-size: 10pt;">In this experiment, the protein gbr22 that was overexpressed in //E. coli// was purified and analyzed. The wet pellet weight for gbr22 was .36 grams. The concentration of gbr22 was 1.18 x 10-3 mg/mL at maximal wavelength. The purity of the protein was about 20% and its MW was about 25kDa. <span style="font-family: Arial,sans-serif; font-size: 10pt;">Future directions in research include purifying gbr22 further using fast protein liquid chromatography as well as considering gbr22 as a protein target and running virtual screening jobs to determine which ligands bind best to the substrate.
 * __ Conclusions: __**


 * __ References: __**
 * 1) Protein production and purification. //Nat Methods.// **2008**, 5(2), 135-146.
 * 2) European Molecular Biology Laboratory. Protein Expression and Purification Core Facility. [] (accessed 4/16/2012).