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  1. 1. Crystallization of Chromatographically Purified 6X-His-tag Green Fluorescent Protein via Induction of pET21b Plasmid from Transformed BL21(DE3) Escherichia coli. Cameron Naglieri-Prescod Biochemistry 276 05/03/2013
  2. 2. 2 Abstract Green Fluorescent Protein is a very useful and interesting tool for scientists to express genes and proteins, and visualize observations of those proteins. In this study, goals of the experiment were to amass a substantial amount of 6X-His-tag GFP, and to crystallize the GFP. The 6X-His-tag GFP gene in a BL21(DE3) E.coli bacterial culture was induced to produce GFP. Via cell lysis, nickel affinity chromatography and hydrophobic interaction chromatography, the GFP was obtained in a purified form. Different solutions from the chromatography methods were exposed to a Bradford Assay and a Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE) to quantitatively determine the concentrations and amounts of total protein in different solutions, and to determine if there is GFP present and the size of the GFP in the solutions. A standard curve and best fit line were obtained via the Bradford Assay. Hanging drop vapor diffusion crystallization of the purest GFP solution was performed with a lysozyme crystal comparison, and the superlative crystals of both the lysozyme and GFP achieved were photographed and observed.
  3. 3. 3 Introduction In the world's oceans, there are numerous creatures that exhibit bright and fascinating colors and patterns. These creatures use these colors and patterns for a multitude of tasks, from finding a mate, to camouflage, to an indicator of danger. There is one creature that uses color in a slightly different manner. The crystal jellyfish, also known as Aequorea victoria, is a simplistic and delicate jellyfish that uses bioluminescence to glow and produce light. What makes this bioluminescence possible is a small protein appropriately named Green Fluorescent Protein, or GFP, because of the emission of green light this protein exerts at a certain wavelength. GFP is used in the biochemical field as a fascinating indicator for the expression of genes and also other proteins. Scientists now are creating perceivable three-dimensional structures of proteins to identify the properties and functions of these proteins at a level smaller than the human eye can see. A set of laboratory experiments were conducted to extract green fluorescent protein from Escherichia coli (E.coli) that has been genetically modified to produce abundant amounts of whatever protein desired in a controlled setting. The main goals of the experiment were to amass a substantial quantity of green fluorescent protein at a high concentration, and to ultimately crystallize the extracted GFP. The E.coli used for the experiment, named BL21(DE3), was engineered to produce a large amount of protein. The engineered plasmid used, named pET21b, contains a T7 viral promoter in its sequence, and this promoter is transcribed by a T7 RNA polymerase; the polymerase is specific to the T7 promoter and therefore will not transcribe any other sequence but that promoter sequence. While the T7 promoter is influenced by the T7 RNA polymerase, the polymerase is controlled by an inducible bacterial promoter, specifically for the lac operon gene on pET21b. The inducer for the bacterial promoter is isopropylthiogalactopyranoside (IPTG). This compound behaves much like lactose in terms of expression of genes, but it is not metabolized so it remains intact. The T7 promoter on pET21b resides next to the multiple cloning site, which in turn resides by the sequence for a specific GFP gene, 6X-His-tag GFP (GFP with six histidine residues attached). The result of specificity in sequencing is a large amount of mRNA, which leads to an immense amount of GFP translated. Induction of the GFP gene was performed on a colony of BL21(DE3) to express the GFP gene in the pET21b plasmid. Cell lysis was performed on the sample in order to extract the induced 6X-His-tag GFP from the plasmids to continue with the purification processes and crystallization; a cell pellet and a supernatant (cleared lysate) were obtained via lysis. Two different methods of purification were implemented to expel the contaminant proteins and to extract a sample of pure GFP. The first method used was Nickel Affinity Chromatography. This method uses a molecule called imidazole, which binds to the 6X-His-tag GFP and keeps it from being washed out of solution. This was used to purify the GFP that was contained in the supernatant that resulted from the cell lysis. Different concentrations of imidazole allowed for higher purification, but lower concentration of GFP. To further purify and to concentrate the sample, Hydrophobic Interaction Chromatography was utilized. This method is based on the idea of salting out proteins. The step adds a high concentration of salt to make
  4. 4. 4 proteins insoluble, and these proteins are then discarded when the salt concentration is changed. In this case, the salt concentration is set high enough so that contaminants are eliminated from solution but the GFP remains in solution. Both the nickel affinity and the hydrophobic interaction chromatography steps purify the specific target protein and concentrate it as well. Qualitatively the chromatography steps show a highly concentrated GFP sample, but quantitatively there is not enough information. Direct Ultraviolet spectrophotometry of protein samples is a widely used method as an estimator for protein concentration, but it does not pinpoint the exact amount. To obtain a direct numerical concentration, a Bradford Assay was conducted. This procedure uses the binding properties of Coomassie Brilliant Blue reagent to bind to protein in order to determine a concentration of total protein in a sample; Coomassie binds to all of the proteins in the sample, so it is not possible to distinguish between the proteins. The protein bovine serum albumin was used in this assay as the control for the standard curve. This assay lacks the capability of distinction of individual proteins in a sample, but does provide an average optical density (OD) of the sample. The information obtained is used to create a standard curve and a linear range of the sample, and this curve allows for the comparison between absorbance value and protein concentration. In order to determine the purity of the GFP sample, a Sodium Dodecyl Sulfate- Polyacrylamide Gel Electrophoresis (SDS-PAGE) was conducted. The basic concept of electrophoresis is the progression of charged particles exposed to an electric field. The SDS- PAGE tracks the mobility of the target protein by denaturing the protein into a primary structure. The negatively charged sodium dodecyl sulfate disrupts the tertiary and quaternary structures of proteins, and when the protein is exposed to a positive charge, the protein has an overall negative charge due to the SDS. When the protein is inserted into the gel, and when a positive electric field is passing through, the protein will drift towards the positive terminus at a rate that corresponds to their molecular weight; a blue dye is inserted to track the protein migration. The result of the SDS-PAGE procedure is distinct bands of protein based on molecular weight. The experiment concludes with the crystallization of the 6X-His-tag GFP. Crystallization requires the protein to have a high concentration to ensure protein growth, but also the right amount of a precipitant to lower the overall solubility of the protein. One of the most common methods of crystallization is the hanging drop vapor diffusion method, which utilizes evaporation. The protein is mixed with a precipitant in a certain ratio, sealed in a container, and left alone to crystallize. Through vaporization and condensation of the precipitant, the crystalline structure of the protein grows while the concentration of the protein increases. The end result is an array of different protein crystals for observation. Based on the various types of purification and testing for protein concentration, a large quantity of 6X-His-tag GFP in a very high concentration is expected to be acquired as a product of the entire procedure, and a large amount of high-concentrated crystallized GFP is expected to result as well from the crystallization method used in the experiment.
  5. 5. 5 Materials and Methods Bacterial Strain and Reagents The bacterial strain used for the experiment was a pET system host strain called Escherichia coli BL21 (DE3). The genotype of Bl21(DE3) is F-ompT hsdSB (rB- mB-) gal dcm (DE3). The encoding of the T7 RNA polymerase gene and the integration of the DE3 lysogen which also contained T7 RNA polymerase gene under the influence of a lactose- inducible lacUV5 promoter on this specific BL21 strain, allowed for the high expression of a target protein, in this case Green Fluorescent Protein (GFP), in an a posteriori manner. The plasmids used for this experiment were pET plasmids that contained the GFP gene, which encoded for the production of Green Fluorescent Protein. The plasmid that contained the GFP gene, called pGFPstop, was a gift from Professor Jeanne Hardy, Department of Chemistry, University of Massachusetts. The GFP gene was subcloned into the multiple cloning site pET21b, which was obtained from Novagen located in Darmstadt, Germany, the coding region of GFP was presented in the most accurate reading frame, and GFP was fused with a 6X His tag located at the C-terminus of that GFP. All of the reagents and supplies needed for this experiment were acquired from Fisher Scientific (Fair Lawn, New Jersey), unless stated otherwise. Induction of GFP expression A group of BL21(DE3) that was transformed with pETHis GFP was used by the teaching staff to inoculate a 5 mL culture of LB medium that contained 100 g/mL ampicillin antibiotic (5 L of a 100 mg/mL stock in 5 mL of medium). The culture