IMRAN+Z

= April 17th, 2013 =

=** An excursion following the gbr22 protein of the //Escherichia coli// bacteria through the processes of expression, purification, and characterization **=

__ Introduction: __

To fully understand the entirety of the experiment involving expression, purification, and characterization of the targeted protein, it is crucial to first understand the meanings of those methods in context. The main objective of recombinant protein expression is to increase the number of copies of the gene or increase the binding strength of the promoter region to assist transcription [1]. This process is commonly used as a pretext to measuring the abundance of a specific protein in a given population of cells. The purification process consists of a series of sub-processes intended to isolate a single type of protein from a mixture containing various forms and formations of molecular matter via chemical and physical filters. A specific type of purification, analytical purification, can be used to produce a relatively small amount of protein to be used for identification, quantification, or modification [1]. In context, protein characterization is a method used for analyzing proteins based on their size. Sieving, the process of applying an electric field to separate charged molecules, such as proteins, is essential for such separation [2]. With regard to tools for analysis, the combination of these methods is essential when selecting a protein to be tested in the virtual environment based off its behavior and characteristics in the laboratory. The overarching goal of the experiment was to utilize specific expression, purification, and characterization methods to target a protein in a single competent cell strain, quarantine it from all other molecular matter in its environment, and characterize it based off of its molecular weight. It was expected that if the protein was successfully harvested and purified, then the characterization process would yield information on the weight of the protein with a high level of precision.

__ Materials & Methods: __

//Protein Expression//

The expression process was initiated by targeting the gbr22 protein housed within the //Escherichia coli // (BL21(DE3)) bacterial species which had been treated with the pGEM-gbr22 plasmid such that it would remain alive in an ampicillin-infused agar plate. After heat shocking the starter-culture tube for 45 seconds and 45OC to promote plasmid uptake, SOC media was added and the tube was placed in a shaking incubator at 37oC. The bacterial solution was added to the agar plate and collirollers were used to spread it throughout the surface. Finally, another dose of SOC was applied and the competent cells were placed in an incubator at 37oC and allowed to mature overnight. A sample of the competent bacterial cells was taken, placed in LB/ampicillin media, and allowed to grow in a shaking incubator at 37oC. The larger culture was created by adding a .625 ml sample from the starter culture to a 25 ml LB/ampicillin broth, which was allowed to grow for 48 hours in a shaking incubator at 37oC. Once the media had turned purple, the first protein sample was taken and the cells were harvested by centrifuging, decanting the fluid, and saving the pellet, which was weighed. The cells were then resuspended in a 1X PBS solution to which stock lysozyme was added and the tube was stored at -20OC.

//Protein Purification//

The purification process was initiated by incubating the solution at room temperature while simultaneously creating 10 ml wash and elution buffers by combining 1xPBS with 20 mM and 250 mM imidazole, respectively. The incubation process was followed by the addition of Benzonase, after which it was again incubated at room temperature. The resulting lysate was dispensed into microcentrifuge tubes and centrifuged. After the second protein sample had been acquired, the supernatant from the tubes was transferred to a conical tube and the lysate was syringe-filtered. .5 ml of Ni-NTA mix was added to the tube and incubated at room temperature while a chromatography column was set-up and rinsed with nanopure water. After adding the mixture to the column, the flow-through was collected as the third protein sample and was flushed once with the wash buffer, the resulting waste being collected as the fourth protein sample. 5 ml of the elution buffer was flowed through the column twice, the first run being collected as “Elution 1” and sample 5 and the second as “Elution 2” and sample 6. The Ni-NTA was stripped by washing the column with 10 cv water, 10 cv .5 M NaOH, and again by 10 cv water, after which 1 ml of 30% ethanol was added and the column was stored. A Nanodrop spectrophotometry machine was cleansed with 2 µl of nanopure water and blanked with the elution buffer. The absorbance of “Elution 1” was analyzed in theA280 mode at 280 nm over two trials. The same process was repeated for the maximal absorption wavelength, 574 nm, in the UV/VIS mode. The wavelengths and absorbances were used to determine and compare protein concentration and yield.

//Protein Characterization//

The characterization process was initiated by utilizing the six samples collected throughout the preceding lab procedures. Sample 1 was centrifuged and the loading buffer was mixed into the remaining pellet. Samples 2-6 were mixed with 6X loading buffer, and all samples were heat-blocked at 95 ° C for 5 minutes, after which they were centrifuged. The electrophoresis module was assembled and a precast gel cassette was used. Each of the ten wells in the gel was cleared with TGS buffer using a needle and syringe, after which the MW standard was loaded into the first well. The subsequent wells were loaded with samples 2-6 in numerical order, and the final three wells were loaded with colleagues’ samples 1, 5, and 6, respectively. The electrophoresis process was initiated and allowed to run for 1.5 hours. The gel was removed, placed in a dish, and washed three times with nanopure water. Imperial stain was added to the dish, which was placed in the imperial shaker for 2 hours. After the gel was destained with nanopure water and a kimwipe had been placed in the dish with water, it was allowed to sit overnight. The gel was then dried at 75 ° C for 2 hours.

__ Results: __



The transparency of the elutions indicates that a very small amount of protein is present in either tube. A = ebc -.37 = (1 cm)(39100)(c) c = -9.46e-6 mol/L (-9.46e-6 mol/ 1 L) * (25794.2 g / 1 mol) * (1 L / 1000 ml) * (1000 mg / 1 g) = -.244 mg/ml Yield = (-.244 mg/ml) * (5 ml) = -1.22 mg This concentration signifies the difference in particulate concentration between Elution 1 and the Elution buffer, the buffer having more material. A = ebc  (.025)(10) = (1 cm)(118300)(c)   c = 2.11e-6 mol/L   (2.11e-6 mol/ 1 L) * (25794.2 g / 1 mol) * (1 L / 1000 ml) * (1000 mg / 1 g) = .054 mg/ml   Yield = (.054 mg/ml) * (5 ml) = .273 mg   The absorbance at the maximal wavelength is higher than the absorbance at 280 nm, as expected. From Sample 5 (Well 6), it can be determined that the approximate molecular weight of the protein is 25,000 Da, compared to 25,794.2 Da as the true molecular weight.

__ Discussion: __

//Protein Expression//

The results of the expression procedure indicated that the pGEM-gbr22 DNA plasmid could be used to alter the //E. coli// bacteria such that the recombinant gbr22 protein would be expressed more potently. During the initial steps of the procedure, the technique of heat-shocking the tube was significant because it promoted the creation of pores in the competent cells’ membranes through which the DNA would be able to traverse to the interior of the cells. The use of collirollers promoted the even spread of the bacterial/SOC mixture without puncturing or aggravating the agar surface. The primary purpose for the addition of ampicillin was to promote selection for only those cells that had absorbed the plasmid, the //E. coli//. It was ensured that the protein was extracted at a point when it was expressed the most strongly, so the bacterial colonies were harvested at some point during the middle of their logarithmic growth phase. Sample 1 consisted of the protein within the //E. coli// cells. Lysozyme was added before storing the cells at a low temperature to ensure that the bacterial cell wall would remain traversable by the protein. Potential errors during this procedure may have been related to the extended time period for which the flask containing the large culture was stored at 4OC – roughly 48 hours – which was longer than planned.

//Protein Purification//

Benzonase was added to reduce the viscosity by digesting the DNA and RNA in the mixture. Sample 2 consisted of all the proteins within the //E.coli// cells as well as small particulates. The Ni2+ in the Ni-NTA mix was used as a binding agent for the HIS tags attached to the end of the negatively-charged protein. This allowed for the creation of a larger molecule that would be prevented from flowing through the column. Sample 3 consisted only of all proteins found in the //E.coli// cells. Sample 4 consisted only of proteins that were loosely bound to the Ni-NTA resin. Sample 5 should have consisted largely of the gbr22 protein, but consisted minimally of the gbr22 protein and mostly of the buffer and any remaining loosely-bound proteins. Sample 6 should have consisted of any remaining gbr22 protein, but was similar to Sample 5 in composition.

The absorbance values at 280 nm and 574 nm were either very small or negative, which was not to be expected. The source of error regarding the negative and minute absorbance values noted in Figure 8 and Figure 9 was, as later discovered, due to a miscalculation of the amount of imidazole to be used to create the elution buffer, which was to have 10X as much imidazole as the Wash buffer, but where a required 2.5 ml was miscalculated to be .25 ml. Due to the small amounts of imidazole added to both the wash and elution buffers, the majority of the gbr22 protein was never stripped from the Ni-NTA in the column, explaining why both the collected Elution 1 and Elution 2 liquids were virtually transparent. Additionally, the original elution buffer used was depleted throughout the course of the experiment, so a buffer produced later was used during the spectrophotometry procedure. This buffer, prepared with the correct amount of imidazole, contained more particulate matter than the original buffer, which is why the concentrations were perceived by the spectrophotometer to be negative – the elution buffer used to blank the machine contained more material than the actual elution. The data from the nanodrop results did, however, signify that absorbancy was higher at the maximal wavelength, which was to be expected.

//Protein Characterization//

The overarching goal of the gel electrophoresis process was to characterize the previously purified protein based off of its molecular weight. Although this was accomplished successfully, the results were not as precise as expected. The presence of excess untargeted proteins in Samples 5 and 6 seen through excess bands in Wells 6 and 7 reaffirmed the power of the previously discovered error regarding an incorrect amount of imidazole used in the elution buffer. Because the buffer failed to extract much of the targeted or even moderately strong proteins from the column, Elution 1 and 2 were composed largely of bits of the targeted protein and many weaker proteins. Because there are two other bands present in Sample 5 other than the targeted protein and they are both of equal intensity to the targeted band, the purity of the protein sample was expected to be roughly 25%. From the band in Sample 5 (Well 6), it can be determined that the approximate molecular weight of the protein is 25,000 Da, compared to 25,794.2 Da as the true molecular weight, a difference of 794.2 Da. This difference outlines the level of precision of the technique used to calculate the true weight compared to the level of precision exercised by the gel electrophoresis process.

__ Conclusions: __

Throughout the breadth of the experiment, the gbr22 protein housed within the //Escherichia coli// stain of competent bacterial cells was targeted. The cells were infused with the pGEM-gbr22 plasmid and over-expressed, purified using various physical and chemical filtration techniques, and characterized using the gel electrophoresis process. Key discoveries included the realization of the crucial importance of the elution and wash buffers, the chemical and physical implications of Ni-NTA purification and gel electrophoresis, the use of Nanodrop spectrophotometry to quantify the viability of the purification process, and finally, the use of a molar weight standard coupled with the process of gel electrophoresis to determine the molecular weight of the protein. At the end of the experiment, it was noted that although the gel was able to provide a rough estimate of the true weight of the targeted protein, the level of precision left a lot to be desired. Purification techniques such as FPLC may be used for future purification purposes, as it would bound the exact weight of the protein to a smaller range and would, therefore, increase the precision of the characterization process and overall experiment.

__ References: __

1. Nordlund, P.; Weigelt, J.; Hallberg, B.M.; Bray, J., Protein production and purification. //Nature Methods//**2008,** 5(2), 135-46. 2. Brody, J.R.; Kern, S.E., History and principles of conductive media for standard DNA electrophoresis. //Annals of Biochemistry//**2004,** 333(1), 1-13.