PRIYA+P

Improve references __ Title: __ Expression, Purification, and Characterization of Purple Fluorescent protein gbr22 in competent E. //coli// cells

__ Introduction: __ Protein over expression serves the purpose of harvesting a large amount of protein that would normally be found in lover concentrations in a native host. The advantage of this is a larger sample to conduct experiments regarding the effect of different ligand docking and binding among other things. For each protein to be expressed, a different type of host cell such has bacteria or yeast, and strain must be uniquely decided [1]. Each protein has a different optimal environment in which it thrives. After expression, the protein must be purified so that only the desired protein remains. Purification techniques have been developed to provide a mix of best purity combined with ease, speed, cost, and versatility [1]. Bl21(DE3), a strain of E. //coli// is suitable host cell to use for producing a large amount of protein [1]. Ampicillin is used to ensure that only cells containing the plasmid grow in culture. Different resins are used to tag the desired protein and separate it from excessive waste. The ratio of column size and recombinant protein can also be adjusted to optimize the purification of protein sample [1]. Protein expression, purification, characterization, and later assay is used to discover new treatments and medicines for a variety of diseases. Therefore fast, efficient, and malleable methods are needed to speed up the long process of drug discovery. The Parallel Sequence and Ligation Independent Cloning method provides this [2]. SLIC joins vectors and inserts on both ends of DNA through homologous  recombination [2] and has no gene size restriction.

The objective of this lab is to over express the protein gbr22 in E. //coli// cells. The protein gbr22 is found in coral from the Great Barrier Reef and causes the host to be fluorescent purple. After expression, the protein will be purified and the characterized to determine the purity and yield levels from the sample. The result of this process will be a high level of purification for the gbr22 protein.

__Materials and Methods__ A plasmid containing the recombinant protein, gbr22, was over-expressed in a lab strain of E. //coli //(BL21(DE3)) //. // The competent bacterial cells were transformed with a DNA plasmid that contained gbr22 and an ampicillin resistance gene to ensure that only bacterial cells that had taken in the plasmid successfully were present. Good antiseptic technique was used and verified through the use of a control plate. A starter culture was grown and used to propagate a larger colony which was harvested and frozen. Sample 1 was taken from the LB + Amp Media containing the gbr22 protein. The bacterial cells were then broken open through the use of a lysosome. All soluble matter from the cells was released and separated from the insoluble matter by centrifugation. Insoluble debris was removed and the chosen protein (gbr22) was tagged with Ni-NTA resin. After purification, the chosen protein resided in Elutions 1 and 2. Samples of Elutions 1 and 2 were analyzed through spectroscopy. Sample 2 was taken from the soluble cell debris after centrifugation. Sample 3 consisted of the flow through waste and Sample 4 of the wash from the purification process. Samples 5 and 6 were taken from Elutions 1 and 2 respectively. Gel electrophoresis (SDS-PAGE) was used to analyze all samples taken from expression and purification. The purity and yield of the protein was determined and correlated to the spectroscopy results. Lab coats, gloves, and protective eye wear was used consistently throughout the process.

__Results__











A=ebc Elution 1 280 nm (.44)(10)=(38850)(1)(c) c= 3.73x10-5 Elution 1 280 nm yield: 5.66x10-5 Elution 1 574 nm yield: 3.06x10-5 Elution 2 280 nm yield: 1.12x10-4 Elution 2 574 nm yield: 3.59x10-6



__ Discussion: __ The protein gbr22 is apparent in samples 5 and 6 of Elutions 1 and 2. Sample 1 was taken from the media containing gbr22, Sample 2 of the supernatant, Sample 3 of waste, and Sample 4 of wash. One band of DNA should be present in each well; however 2 bands and the blot are visible in each. The protein gb22 was determined to be the third band from the well label since it was absent in samples of the waste and wash. The protein is absent from these two samples because the HIS tag serves to bind the protein to the Ni-NTA resin/buffer mix. The protein then is bound to the nickel on the beads and cannot flow through while all other proteins and matter without the tag flow through. Wash Buffer containing imidazole was used to strip the proteins that were weakly bound to the resin. Imidazole competes with the HIS tag, causing the protein to be stripped. The Elution Buffer with a high concentration of Imidazole compared to the Wash Buffer was used to strip the tightly bound gbr22 protein into Elutions 1 and 2. Lysosome was used to break open the cell wall of the bacterial cells to release all soluble matter into solution. The significance of this process is to be able to separate the chosen protein from all other soluble and insoluble waster after centrifugation and the purification process. Cyanase was used to digest any RNA/DNA that had been released into solution. All that should have remained in Elutions 1-2 should have been gbr22 protein, but another protein was not stripped of the tag. This error could stem from the Elution Buffer not having a strong enough concentration or contamination with the original plasmid used. The purity of gbr22 is only about 40% since 2 other bands are present Sample 5 well. 1 band is at the same intensity as gbr22 and the other is considerably lighter. The size of protein determined by the Nano-drop is .42 mg/ml at 280nm. This corresponds with results from the gel. The Nano-drop results determine how much of a protein is present and the gel helps determine the purity of the sample.

__ Conclusions: __ The protein gbr22 was expressed, purified, and then characterized. The purity of the sample was about 40% and there was about .42 mg/ml at 280nm. The sample was contaminated and was therefore not at a high purity level. The next steps would be to complete protein assay. This process will be used in the future when a specific target protein from a viral disease is chosen.

__ References: __ 1. Nat Methods**. 2008** Feb;5(2):135-46. Protein production and purification.

2. BMC Biotechnol. **2013** Feb 14;13:12. doi: 10.1186/1472-6750-13-12.