LukeP

= = =**Luke P.** = =Expression, Purification, and Characterization of the GBR22 Purple Protein=

__**Introduction and Objective:**__ With the widespread expansion of technology, the method for producing, purifying, and characterizing recombinant proteins is now relatively simple and cheap to conduct in the lab. Recombinant proteins can then be purified and studied to develop therapies for certain medical conditions or to contribute additional knowledge of the protein to further research [4].

In this experiment, a plasmid called pGEM-gbr22 was cloned into the //Escherichia coli// bacteria’s genome. After taking up the plasmid, the bacteria could then express the gene for the gbr22 protein in a culture. Also in the plasmid was encoded a resistance to the antibiotic, ampicillin, with a beta lactamase enzyme capable of destroying the drug. Therefore, only the transformed bacteria could grow on the cultures containing the antibiotic. In this experiment, the recombinant protein expressed was a purple protein derived from a species of coral in the Great Barrier Reef with an affinity tag attached. The affinity tag of six histidine residues permitted the protein to be isolated quickly and effectively during purification by adhering to the nickel cations in the agarose. In addition, the affinity tag would “rarely adversely affect biological or biochemical activity” of the protein [4].

After lysing the bacterial cells and removing large and insoluble cell debris, chromatography with a nitriloacetate agarose (Ni-NTA agarose) was performed to purify the protein. The affinity tag of six histidines could bind to the nickel cations, thus isolating the protein from the rest of the cellular material contained in the E. coli bacteria. After releasing the protein from the Ni-NTA agarose with Imidazole, which competed with the protein in binding to the nickel ion, the protein was purified. Using the spectrophotometer, Beer’s law, and data collected regarding the purple protein's properties, the concentration of the purified protein in solution was calculated.

In order to characterize the protein’s purity and molecular weight, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the proteins from each step in purifying the protein. Before electrophoresis, the protein samples were exposed to SDS to denature every protein and apply a negative charge to the proteins relatively to their molecular mass. The samples were then placed in the gel and separated by their charge to mass ratio. Since the charge at the bottom of the gel was positive, the negatively charged proteins were pulled down through the gel. Purification prevents the wasting of “resources on protein material of inadequate quality” and “to ensure that different batches of the same protein have similar properties” [4]. Therefore, the purified protein should be devoid of any other proteins that could interfere with research, and the sample can be compared to others to verify the correct protein was isolated.

The objective of this lab was to express the purple protein (gbr22) in E. coli cells, purify the protein using the Ni-NTA agarose in the chromatography column, and then characterize the protein through gel electrophoresis. The techniques developed through this lab can be used to express, isolate, and characterize virtually any target protein responsible for a disease that could be tested in virtual drug screening. After potential ligands are determined from virtual screening, the ligands could be purchased and their effects tested on the purified target proteins.

__**Materials and Methods:**__ In each of the three labs, appropriate safety gear, such as goggles and a lab apron, were worn to prevent contamination with samples and to protect body tissues from any chemicals or organic used. Also, all waste generated throughout the lab was disposed of in the proper receptacles.

__**Expression:**__ Three cultures were prepared: an experimental plate,a control plate, and a “fun” plate. Two sets of properly prepared New England BioLab BL21(DE3) competent E. coli cells were produced. The control was left alone, and the other was exposed to the pGEM-gbr22 plasmid carrying the gene for the gbr22 protein and ampicillin resistance. The two samples were plated in an ampicillin containing medium along with SOC media. The “fun” plate without the antibiotic was coughed on, and all three plates were placed in the incubator overnight. The purple colonies of transformed bacteria were transferred to a liquid starter culture containing ampicillin and LB media, and incubated in the shaker for several hours. The culture was then transferred to a larger liquid culture with ampicillin and fresh LB for overnight expression. The cells were harvested in a pellet by centrifugation after collecting Sample 1 for the protein characterization portion of the lab ,and then suspended in phosphate buffered saline with a lysozyme before freezing.

__**Purification:**__ The transformed cells were lysed with the added lysozyme, and then Benzonase was added to reduce the viscosity. The sample was then clarified with the centrifuge and syringe filter to remove insoluble and large cellular debris. After centrifugation, Sample 2 was collected. The protein was then exposed to the Ni-NTA agarose and placed in a chromatography tube with PBS. The waste was collected as Sample 3, and the column was washed with a low concentration of imidazole and PBS. Sample 4 was taken from the wash. Elutions 1 and 2 were then collected in consecutive collections after exposure to a higher concentration of imidazole and PBS. Elutions 1 and 2 were then used as Samples 5 and 6 respectively. Each elution was then analyzed with the Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) at two different wavelengths.

__**Characterization:**__ Sample 1 was centrifuged, and the pellet was isolated and resuspended. The Mini-PROTEAN electrophoresis tank was assembled and filled with TGS buffer. Each sample mixed with the loading buffer was placed in one of the Bio-Rad precast polyacrylamide gel’s wells along with the Fermentas PageRuler PreStained Protein Ladder. After running electricity through the gel, it was then stained, destained overnight, and dried.

__**Results:**__ The following three images depict the plate with the transformed bacterial colonies, the negative control plate of bacteria without the plasmid, and the “fun” plate that was coughed on.





T he plate with the transformed bacteria contained approximately twelve purple colonies. The negative control plate exhibited no growth since the bacteria was not resistant to the ampicillin.

<span style="font: normal normal normal 12px/normal Helvetica; letter-spacing: 0px; margin: 0px;">The following images show the large cultures after overnight expression and the cell pellets harvested from the cultures. <span style="font: normal normal normal 12px/normal Helvetica; letter-spacing: 0px; margin: 0px;">The following picture depicts the first and second elutions from purifying the protein in the chromatography column. = = <span style="font: normal normal normal 12px/normal Helvetica; letter-spacing: 0px; margin: 0px;">The next four absorption spectrums were measured from the first and second elutions. The first two absorbances were taken at 280 nanometers with the spectrophotometer, while the last two absorbances were measured at the maximum absorption for the gbr22 protein, 574 nanometers. The maximum absorption wavelength and extinction coefficient ( 118300 liters per mole times centimeters) at that wavelength were found on the cited article under the chromoprotein heading for //Montipora efflorescens// (the coral the purple protein came from) [1]. In addition, the extinction coefficient at 280 nanometers (38850 liters per mole times centimeters) for the protein and its molecular weight (25794.2 grams per mole) were calculated using an online tool [3]. <span style="font: normal normal normal 12px/normal Helvetica; letter-spacing: 0px; margin: 0px;">Using Beer’s law, the concentration and yield of the purified protein from the first elution at each wavelength was calculated.

<span style="font: normal normal normal 12px/normal Helvetica; letter-spacing: 0px; margin: 0px;">According to Beer’s law, absorbance (A) is equal to the product of the extinction coefficient, or molar absorptivity (E), in liters per mole times centimeters, the concentration of the solution in moles per liter (b), and the path length of the cuvette used in the spectrophotometer (c). Therefore, b = A/(Eb). At the 280 nanometer wavelength, the average absorbance was .0665, so the concentration is equal to (.0665)/(38850*1), or 0.000001712 moles per liter. Multiplying that concentration by the molecular weight of the protein (25794.2 grams per mole) gives the concentration of the protein as .0042 milligrams per milliliter. Multiplying this value by the volume of Elution 1 (4.5 mL) gives the protein’s yield as .199 milligrams. At the maximum absorption wavelength, the average absorbance was .215. Therefore, the concentration equaled (.215)/(118300*1), or 0.000001817 moles per liter. Multiplying that concentration by the molecular weight gave the final concentration of .0469 milligrams per milliliter. By multiplying the concentration by the volume of Elution 1, the protein’s yield was .211 milligrams.

<span style="font: normal normal normal 12px/normal Helvetica; letter-spacing: 0px; margin: 0px;">The following images show the completed gel before and after drying, and the protein ladder used in this experiment.

<span style="font: normal normal normal 12px/normal Helvetica; letter-spacing: 0px; margin: 0px;"> <span style="font: normal normal normal 12px/normal Helvetica; letter-spacing: 0px; margin: 0px;"> <span style="font: normal normal normal 12px/normal Helvetica; letter-spacing: 0px; line-height: 19px; margin: 0px;">Figure 7C: The molecular weight of each band for the PageRuler PreStained Protein Ladder is displayed [2]. The purified protein aligned with the third band from the bottom of the ladder giving a molecular weight near 25 kiloDaltons.

Comparing the purified protein to the protein’s ladder on the gels made in this lab, the protein aligned with the third marker from the bottom of the ladder. Using the image for the ladder from the manufacturer, the purified protein should have a molecular weight near 25 kiloDaltons. In addition, the wells in the gel containing the two elutions had only trace amounts of any other bands. Therefore, the protein was approximately 99 percent pure.

<span style="font: normal normal normal 12px/normal Helvetica; letter-spacing: 0px; margin: 0px;">__**Discussion:**__ <span style="font: normal normal normal 12px/normal Helvetica; letter-spacing: 0px; margin: 0px;">The plasmid successfully transformed the bacteria which then expressed the purple protein and ampicillin resistance. The negative control of the plate of bacteria without the plasmid exhibited no growth, proving the techniques used in the lab were sterile. From the transformed bacteria, the cells were cultured and later harvested, and then purified using the Ni-NTA agarose in the chromatography column. The concentration and yield at each wavelength was then calculated. Since the two values for the concentration were less than .003 milligrams per milliliter apart and the two calculated yields were only .012 milligrams apart, the results were extremely precise and most likely accurate.

<span style="font: normal normal normal 12px/normal Helvetica; letter-spacing: 0px; margin: 0px;">Each step of the purification process was processed through gel electrophoresis correctly. The second l ane containing Sample 1 came from the completed cell culture following protein expression, so the lane contains multiple proteins used by the E. coli. Lane 3 contains the clarified cell lysate with an abundance of proteins in a higher concentration than Lane 1 since the cells had been lysed with the internal proteins exposed. Lane 4 was the waste from the first flow through with the chromatography tube, so it contains most of the proteins that did not bind to nickel cations in the agarose. Lane 5 was from the wash through, further purifying the purple protein. The lane contains the remaining proteins that were removed and isolated from the Ni-NTA agarose. The last two lanes contain each elution, with the first elution containing the majority of the purified protein, and the second containing very little of the purple protein. Given that the actual molecular weight of the protein was found to be 25.79 kiloDaltons and the protein ladder reference estimated the molecular mass as 25 kiloDaltons, the protein was successfully purified. Since the two molecular masses were so near each other, the identity of the purified protein was verified as the gbr22 protein. In addition, the protein was correctly purified since it contained less than one percent of any contaminants.

<span style="font: normal normal normal 12px/normal Helvetica; letter-spacing: 0px; margin: 0px;">Since there was little evidence to support any imprecision or inaccuracies in each labs, the possibility of error was somewhat slim. While the protein was being expressed in the bacteria, some of the techniques may have been slightly unsterile. While purifying the protein, mistakes made while manipulating the chromatography tube could have caused the presence of any impurities in the elutions. During characterization, the results could have been skewed since the the gel “smiled” slightly. Lastly, any of the solutions used in throughout the lab could have been prepared incorrectly or at the improper concentrations to further deviate the data.

<span style="font: normal normal normal 12px/normal Helvetica; letter-spacing: 0px; margin: 0px;">__**Conclusion:**__ <span style="font: normal normal normal 12px/normal Helvetica; letter-spacing: 0px; margin: 0px;">The method for expressing a target protein in a bacterial culture was developed. The target protein could then be purified using the techniques from the second portion of the lab. Lastly, the target protein could be characterized using gel electrophoresis to ensure that the samples are pure and contain the appropriate protein.

<span style="font: normal normal normal 12px/normal Helvetica; letter-spacing: 0px; margin: 0px;">Concerning virtual screening, a target protein responsible for a certain disease could be expressed in bacteria. The protein could then be isolated from the bacteria, and purified using similar methods discussed in this lab. The target protein could then be characterized using gel electrophoresis to determine the sample’s purity and to verify the protein is actually the target. After virtually screening a library of ligands along with the target protein's crystal structure, the top ranked ligands determined by the computer could be purchased and tested in the wet lab using assays. In this way, novel inhibitors could eventually be developed as new drugs for certain medical conditions.

<span style="font: normal normal normal 12px/normal Helvetica; letter-spacing: 0px; margin: 0px;">__**Works Cited:**__ <span style="font: normal normal normal 12px/normal Helvetica; letter-spacing: 0px; margin: 0px;">1.Alieva, N. O.; Konzen, K. A.; Field, S. F.; Meleshkevitch, E. A.; Hunt, M. E.; Beltran-Ramirez, V.; Miller, D. J.; Wiedenmann, J.; Salih, A.; Matz, M. V., Diversity and evolution of coral fluorescent proteins. PLoS ONE [Online] 2008, 3, (7), e2680. [|__http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2775108/__] (accessed Apr 18, 2011).

<span style="font: normal normal normal 12px/normal Helvetica; letter-spacing: 0px; margin: 0px;">2.PageRuler Prestained Protein Ladder page from Thermo Scientific. [|__http://www.fermentas.com/templates/files/tiny_mce/media_pdf/migration_sm0671_2008.pdf__] (accessed Apr 18, 2011).

<span style="font: normal normal normal 12px/normal Helvetica; letter-spacing: 0px; margin: 0px;">3.ProtParam Tool page from Swiss Institute of Bioinformatics. [|__http://ca.expasy.org/tools/protparam.html__] (accessed Apr 18, 2011).

<span style="font: normal normal normal 12px/normal Helvetica; letter-spacing: 0px; margin: 0px;">4.Structural Genomics Consortium, et al., Protein production and purification. Nat Methods [Online] 2001. 5 (2): 125-46. [|__http://www.nature.com/nmeth/journal/v5/n2/full/nmeth.f.202.html__] (accessed Apr 18, 2011).

<span style="font: normal normal normal 12px/normal Helvetica; letter-spacing: 0px; line-height: 0px; margin: 0px; overflow-x: hidden; overflow-y: hidden;">

<span style="font: normal normal normal 12px/normal Helvetica; letter-spacing: 0px; line-height: 0px; margin: 0px; overflow-x: hidden; overflow-y: hidden;">