KEELY+W

__ Title: Protein production by expression, purification, and characterization of pGEM-gbr22 in //E.coli// __

__ Introduction: __ Protein expression, purification, and characterization of recombinant proteins have been used for protein production for years. However, there are many strategies that need to be taken into consideration such as: affinity tags, selective markers, bacteria hosts, and plasmid strains. The use of the bacteria //E. coli// as the recombinant host produces high-level protein production[1]. Specifically, the BL21(DE3) //E. coli// strain which has a protein that is membrane bound (Not sure if this is relevant), one or more disulfide bonds, and contains additional tRNAs [2].

The techniques: protein expression, purification, and characterization will be used in this lab to extract and characterize an antibiotic resistant protein from the gbr22 protein using the six-histidine affinity tag and BL21 (DE3) competent bacteria cells. From characterization, the molecular weight, purity, and yield will be estimated. It is predicted that the gbr22 protein will be overexpressed in //E.coli//, purified, and characterized with 1 band on the gel electrophoresis. This signifies that the protein is pure and uncontaminated.

(Maybe could add a little more theory behind expression, purification, characterization than was stated)

__ Materials & Methods: __ The pGEM-gbr22 protein, which was already cloned with the gene conferring ampicillin resistance (proteins are not cloned and transformed, DNA is), was transformed with the competent //E. coli// BL21(DE3) cells. Then 25µL of BL21(DE3) and 1-2 µL of plasmid were added to grow //E.coli///BL21(DE3). The next morning, the colonies were inoculated in a media containing 10µL ampicillin, and placed in 37° C 200-350 rpm shaking incubator. After at least 8 hours of incubating, .625mL starter culture was tranferred to a flask with 50µL ampicillin and 25mL of LB, and left to incubate till the media turned purple. A 500µL sample labeled ”Sample 1” was taken and stored in 4° C freezer. The cells were then harvested by centrifuging (What conditions). The pellet on the bottom of the tube was saved and weighed, while the excess liquid was discarded. Then, the cells were re-suspended by adding 2.5mL of 1x PBS and 50µL of lysozyme to the tube with the pellet and vortexed. The pellet was then stored a -20° C freezer to use for purification.

For purification, the suspended pellet was thawed. Then, 1µL of Cyanase was added, incubated, and then centrifuged (Conditions?). After centrifugation, a 50µL sample was taken and labeled as “Sample 2” to store in a 4° C freezer. The liquid supernatant containing the purple protein from centrifugation transferred into a 15mL conical tube. The supernatant was then filtered with a .22 PES syringe filter and a 5mL syringe into a 14mL round bottom transformation tube to get rid of large particulate matter. After filtration, .5mL of Ni-NTA homogenous resin/buffer mix was added. The column chromatography apparatus was set up using a 20mL Bio-Rad chromatography Econo column. The resin and buffer were added to the column and liquid was dispensed through the column, leaving the resin behind. A 50µL sample was collected from this flow through step and labeled as “Sample 3” and stored in the 4° C freezer. Then the Ni-NTA resin was washed with 5mL of 20mM imidazole in 1x PBS, and a 50µL sample was taken and labeled as “Sample 4”. The bound protein was eluted by adding 5mL of the 250mM imidazole (Imidazole is not the only thing in the buffer solution ) and labeled as “Elution 1”. The protein was further eluted by adding another 5mL of elution buffer like in elution, and collected and labeled “Elution 2”. Samples 5 and 6 were collected from transferring 50µL of elution1 and 2, then stored in the 4° C freezer along with elution 1 and 2. A Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) was used to determine the absorbance of the purified protein at 280nm and calculate the yield and concentration of gbr22.

From purification, the protein samples were characterized using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The samples were prepared by adding a 6x gel-loading buffer. Theses samples were heat blocked at 95° C for 5 minutes and centrifuged. For gel electrophoresis, 7µL molecular weight standard PageRuler Prestained Protein Ladder (product#266616, Thermo Scientific, Rockford, IL) was added to the first well, and 20µL of each sample were loaded into the wells. The gel was run, stained with Imperial protein stain, washed, and then dried. (This last sentence needs to have many more descriptions)

__ Results: __

 Fig1a. Bacterial plate with pGEM-gbr22 and BL21 (DE3) on Amp plate after incubating overnight at 37 ° C.

====Fig1b. Control Plate that has no DNA on an Amp plate after incubating overnight at 37 °C. ====  Fig 1c. Fun Plate on Agar with no DNA and no Ampicillin after incubating at 37 °C overnight.

Fig 2. Grownculture expressed in purple after being incubated in the shaking incubator at 37 °C for 16-24 hours. Media contains LB and Ampicillin as well as BL21(DE3) and the DNA pGEM-grbr22.

Fig.3 WetPellet at .37 grams after being centrifruged for 10 minutes at 5,000rpm at 4°C.

Fig.4 5mL of the purified gbr22 protein after washing 1xPBS and 250mM imidazole solution elution buffer. Elution1 is light purple.

Fig.5 5mL of the purified gbr22 protein after washing 1xPBS and 250mM imidazole solution elution buffer. Elution2 appears clear with a hint of purple.

Fig.6 Absorbance vs. wavelength for Elution1(sample 5) using a Nanodrop Spectrophotometer(Thermo Scientific, Wilmington,DE) at 280nm on the "Protein A-280" setting. The absorbance was found to be .087mg/ml.

From the Nanodrop spectrophotometer, Beer’s Law was used to calculate the concentration of purified gbr22. At 280nm wavelength the concentration and yield was calculated to: C= A/(//ε*b) A= (.087+.081)/2 = .084// //b=1// //ε//// = // 38850 The yield of purified gbr22 at its maximal wavelength (574nm) was .12537mg.
 * C**= .084/38850 = 5.58 x 10-2mg/mL
 * Yield:** [5* (58x10-2)] * 5mL = .279mg

Fig. 7 Electrophoresis gel with samples 1-6 before drying covered in saran wrap, the molecular weight standard PageRuler Prestained Protein Ladder (product#266616, Thermo Scientific, Rockford, IL) is represented in lane 1.

Fig.8 Electrophoresis gel with samples 1-6 after drying. Lanes are labeled.The molecular weight standard PageRuler Prestained Protein Ladder (product#266616, Thermo Scientific, Rockford, IL) is represented in lane 1. (Might want to also consider putting the lanes and what's in each in the caption)

Fig.9 Molecular Weight Standard: SDS-PAGE band profile of the Thermo Scientific PageRuler Prestained Protein Ladder(product#26616, Rockford, IL).

__ Discussion: __ The dry gel from Fig.8 displayed the different samples of protein purification. Sample 1 in lane 1 represented the bacteria cells lysed. Sample 2 in lane 3 represented the supernatant after adding the Cyanase enzyme and centrifuging. Sample 3 in lane 4 was the flow through solution, and sample 4 in lane 5 represented the weak wash of 20mM imidazole. Elution1 was displayed in lane 6 and Elution2 in lane 7. Lanes 8-10 represented the additional runs of samples 4-6. The wash (Sample4) in lane 5 was less purified because it added a weaker concentration of imidazole (20mM) than Elution1 in lane 6, which added 250mM imidazole. The weak wash only removed the loosely bound proteins, while the elution had a high enough concentration to release the protein from the Ni-NTA resin altogether. (Talk about competition of imidazole for the Ni-NTA resin)

By using the molecular weight standard shown in Fig. 9, the molecular weight of gbr22 estimated to be about 25kDa. This was similar to the 25794.2 grams found in the purification lab. The wet pellet created from the protein expression lab weighed .37 grams, while the yield from purification calculated to .202185, averaging the yield at 280nm and 574nm. This signified the large, and loose molecules being removed from the wet pellet of the expression lab to a more purified substance. ( ??? )

Purification required for the overexpressed //E.coli// cells to be broken down. The lysozyme broke down the cell walls, allowing the DNA to be released. Then Cyanase was added to digest the DNA/RNA in the mixture and made it more viscous. The six-histidine tag was then introduced by binding its residues to nickel, which immobilized the protein to the Ni-NTA agarose column matrix. A high concentration of imidazole unbinded the histidine residues to the nickel allowed for the remaining purified protein to be released.

The results of protein characterization by gel electrophoresis presented in Fig.8 showed the gbr22 had 3 bands instead of the intended 1 for Elution1.The three bands were equal in there high intensity by its bolded bands. This indicated that the gbr22 had a purity of 33%. The plasmid used in the protein expression lab could have been contaminated. Other possible errors that would affect the results are: unsterile techniques, incorrect buffer concentrations, or error in the stripping process of the column chromatography.

__ Conclusions: __ The pGEM-gbr22 protein was expressed in //E. coli//. The protein was purified by lysing and column chromatography, and then characterized by gel electrophoresis. Protein production and purification are essential processes to testing and analyzing ligands. A protein has to be completely pure in order to effectively test a ligand. Based on the characterization from the gel, the protein had 33% purity, which failed the hypothesis. Because the protein’s lack of purity, further purification techniques are required. After the protein has been completely purified, the protein could be used to test ligands for drug potential.

__ References: __
 * 1) Protein production and purification. Nat Methods **2008**, 5, (2), 135-46.
 * 2) European Molecular Biology Laboratory.Protein Expression and Purification Core Facility. [] (Accessed April 16,2013).
 * 3) Thermo Scientific. PageRuler Prestained Protein Ladder 10-170K. [] (Accessed April 10, 2013).