VICTORIA+G

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Keep title professional

Make intro more relevant to this lab M&M not detailed enough Improve captions Discussion not very profound, weak analysis Flow of discussion hard to follow Improve references

__ Title: __ The Prettiest Protein in the Lab: The Expression, Purification, and Characterization of gbr22.

__ Introduction: __  Producing and purifying a recombinant protein is a delicate task that has gradually become a more common lab practice. It has long been the goal to develop methods of protein expression and purification that are effective in terms of time, cost, and accuracy[1]. Escherichia coli is commonly used as a vector for protein expression, with the strain BL21(DE3) being particularly effective with eukaryotic cells[2]. Ampicillin is the most common antibiotic marker, although carbenicillin has been found to be more stable[2]. Purification is usually accomplished using chromatography after lysing the expressed bacterial cells, and specificity is commonly increased using varying concentrations of imidazole[2]. Although these techniques have been developed over time and are very widely used, there are common errors that occur, including poor lysing techniques, expression of a mutant protein, or the presence of multiple proteins after purification[2]. Even with negative possible outcomes, these strategies are the best option for expressing and purifying a protein.  It is the intention of this lab to express gbr22 protein in Agar plates with BL21(DE3) bacterial cells and ammpicillin. The protein will then be extracted and purified, and the net product will be characterized using electrophoresis. The final result should be a solution containing the single gbr22 protein.

__ Materials & Methods: __ The protein gbr22 was overexpressed using the plasmid pGEM-gbr22 in BL21(DE3) bacteria on LB agar plates with an ampicillin marker. A starter culture was grown and amplified in LB. The resulting cells were centrifuged and harvested as a pellet resuspended in phosphate buffered saline solution, then frozen for future use. The bacterial cells were then lysed. After centrifugation, the insoluble cell remnants were disposed of. The soluble protein was purified in a Bio-Rad Econo chromatography column using an affinity tag (HIS tag) and Ni-NTA resin. Various samples were collected along the way and stored. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to analyze the samples from the protein expression and purification. The electrophoresis and spectroscopy results the molecular weight, purity, and final yield of the protein product. The protein solution concentration was determined by correlating the results to previous spectroscopy data.

__ Results: __ In the Nanodrop spectrophotometry, the following yields were calculated. Elution1, wavelength of 280 nm A=εbc (0.441)(10)=(39100)(1)(c) 4.41=39100c c=0.00011279 cv=yield (0.00011279)(5)=0.00056395 mg yield= 5.6x10 -4

Elution 2, wavelength of 280 nm A=εbc (0.087)(10)=(39100)(1)(c) 0.87=39100c c=0.00002225 cv=yield (0.00002225)(5)=0.00011125 mg yield= 1.1x10 -4

Elution 1, wavelength of 574 nm A=εbc (0.0725)(10)=(118300)(1)(c) 0.725=118300c c=0.00000613 cv=yield (0.00000613)(5)=0.00003065 mg yield= 3.1x10 -5

Elution 2, wavelength of 572 nm A=εbc (0.0085)(10)=(118300)(1)(c) 0.085=118300c c=0.00000072 cv=yield (0.00000072)(5)=0.0000036 mg yield= 3.6x10 -6

__ Discussion: __ Lysozyme was used to break down the peptidogycan in the cell membranes of the BL21(DE3) bacteria so that the protein could be released. Cyanase was used to destroy any DNA and RNA to purify the soluble protein. During purification, a HIS tag system was employed. HIS tags were present in the protein. These bound to the Ni-NTA resin, and prevented the protein from being washed through with unwanted soluble cell debris. Imidazole also contains HIS tags.A low concentration was added to the wash buffer to wash out any extraneous proteins clinging loosely to the resin. Once the soluble protein was to be washed through into purified elutions, the elution buffer, with a much higher concentration of imidazole, was added. The HIS tags in the imidazole competed with those in the protein, and forced the gbr22 out as the imidazole bound to the Ni-NTA resin. Six samples were taken throughout the expression and purification processes. Sample 1 contained the bacterial culture, Sample 2 contained the supernatant after centrifuging the lysed bacterial cells, Sample 3 contained the flow through waste with 1x PBS from the chromatography column, Sample 4 contained the wash buffer with 1x PBS and 20mM imidazole with extraneous soluble materials, Sample 5 contained Elution 1 of the purified protein, and Sample 6 contained Elution 2 of the purified protein. The Elution buffer contained a much higher concentration of imdazole than the wash buffer ( 250mM vs. 20mM). According to spectroscopy, the protein size was 0.47 mg/ml, while the gel revealed a purity of approximately 35%. According to the gel, there were two other proteins present in the final elution product. The one that was bigger than gbr22 made up less of the composition (approximately 30%), while the smaller one made up more of the composition (approximately 35%). There were obviously errors that occurred in this lab. These could have been possible contamination of the DNA plasmid, an error in sterile techniques (i.e. inadequate proximity to the gas burner or unnecessary exposure to air), or various errors in calculating solutions or physically purifying the protein.

__ Conclusions: __ The protein gbr22 was overexpressed in BL21(DE3) bacterial DNA, and cultured. The bacterial cells were then lysed and purified through centrifugation and chromatography. Gel electrophoresis was then used to analyze the purity of the final protein product. The final product was not pure gbr22, but instead consisted of two additional unknown proteins. This could have been due to a number of possible contamination or procedural errors. The skills developed in this lab will serve as the base for future wet lab research as individual research begins and a select protein target is synthesized for testing.

__ References: __ 1) European Molecular Biology Laboratory. Protein Expression and Purification Core Facility. [] (Accessed April 16,  2013). 2) Protein production and purification. Nat. Methods 2008, Feb 5(2), 135-46. 3) Thermo Scientific. PageRuler Prestained Protein Ladder. [] (accessed April 13,  2013).