Shelby+F-G

__ Title: __ Protein Expression, Purification and Characterization: pGEM-gbr22 developed in E. coli

__ Introduction: __ Proteins are ubiquitous in nature, and are the basic composition of all life. New proteins are being discovered continually, and the first questions that must be answered about them are basic questions about their structure and function. Recombinant proteins are of particular importance, and are often used throughout the biological sciences and medical fields. These proteins are not only important, but are necessary to study to continue vital research, and this research would not be possible without first determining some key facts about the recombinant protein. But these simple facts are not necessarily easily ascertained, as the methodology behind finding this information varies widely, and relies on the judgment of the researcher to determine which methods will produce the highest quality and most effective results. One particular process, the one conducted in this experiment, employs the use of bacteria, most typically e. coli, to over express the recombinant protein, which can later be purified and characterized to determine such basic facts as the molecular weight of said protein. The objective of this lab is to over express, purify then characterize the protein pGEM-gbr22. This process will include injecting the protein into e.coli, incubating the bacteria, lysing it open, and harvesting the grown protein to put through electrophoresis in order to determine its molecular weight. The expected outcome is that the bacteria will yield usable cultures that will produce enough protein so that its pure molecular weight will be identifiable next to a ladder.

__ Materials & Methods: __ Using the strain of e. coli identified as BL21 (DE3), the plasmid Pgem-gbr22 was grown through proper incubation on an LB agar plate. This was a four step process over the course of three days which encompassed placing the plasmid on the plates and allowing them to transform overnight, separating off the cultures and growing them as start cultures for eight hours, then growing them as large cultures for another twenty-four hours, followed by harvesting the bacteria. In order to prevent competition with other bacteria, the LB plate had been infused with ampicillin, since the protein of interest was designed to be resistant. Incubation was done in a 37 degree incubator, and ice baths were used in between to put the protein into shock. The sterile technique was also used throughout the process to prevent contamination. Once this over expression process had been completed, a lysozyme was added to start breaking apart the e. coli. The protein was then run through a series of washes, and elutions, with several runs through the centrifuge, the protein was purified, with samples being taken after the washes and elutions to test at a later point. The electrophoresis gel, of a four to twenty percent gradient, was then set up with a reference ladder that ranged from ~10 to ~150, and samples 1-6, and 4-6 of another experimenter’s, were placed on the same gel and the electrophoresis started. Once completed, the gel was repeatedly stained, then cleaned, and dried. Upon completion, the various samples were compared to the ladder to determine the molecular weight of the final protein, or sample five.

__ Results: __ Over the course of the experiment, photos were taken to document the steps, and what the particular protein being worked on looked like at each of these steps. Ultimately, all of these steps produced the gel that indicated a weight of ~25.

IMPROVE CAPTIONS







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__ Discussion: __ The results of this experiment tell not only the predicted molecular weight of this protein, but give an almost quantitative analysis of the effectiveness of the process, and the experimenter conducting it. The percent yield and purity of the protein both speak to this effect, and enable a future experimenter to perhaps note, and improve upon the technique used. Sources of error primarily include the fault of the experimenter, who, on multiple occasions, created discrepancies and errors, some of which were caught and probably several others that were not. Throughout the experiment, major faults included the failure of the bacteria culture to grow, which lead to the experimenter utilizing the bacteria grown by a lab partner, bad injection or staining, which lead to three bands of equal intensity on the gel in lane five, which would suggest the protein was impure, and several instances where supplies from another source had to be utilized because the supplies originally created for this purpose were either used improperly, or could not be found once experimentation had started. For this lab, lysozyme was used to break up the parts of the bacteria that were unnecessary once the protein had been fully developed, so that the protein could be separated out. The HIS tag system works by attaching a long sequence of histadines, which, due to the rarity of such a long strand of the same amino acid occurring, make it stand out, and attach to things it would not otherwise attach to. After sample one, the majority of the bacteria was still intact, by sample two, one wash had been completed, by three, two washes, and so only major components remained, by four, the final wash had taken off the last bits of detached particles, sample five occurred after elution one, which took away most of the remaining non-protein molecules, and by six, the final elution had been completed. The wash and elution buffer differ in concentration of immidazole, which greatly impacts how much and what kind of molecules are being washed away, with the elution containing a large enough amount to knock the protein free. Based on the gel produced from this experiment, the protein was only about 35% pure at that point, and measured ~25.

__ Conclusions: __ This was a three part experiment which covered the expression, purification, and characterization of a protein. The key finding was that the molecular weight was ~25, but the purity was not very good. This could indicate areas of improvement in this type of lab, as well as providing information about this protein.

__ References: __

European Molecular Biology Laboratory. http://www.embl.de/pepcore/pepcore_services/index.html(accessed April 17, 2013). Protein Expression and Purification Core Facility. Genedirex. http://www.genedirex.com/?cat=6(accessed April 17, 2013). Guide for Molecular Weight Estimation image. NCBI. PubMed.org. http://www.ncbi.nlm.nih.gov/pubmed/18235434(accessed April 17, 2013). [|Nat Methods.] 2008 Feb;5(2):135-46. doi: 10.1038/nmeth.f.202.