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__ Title: __ Expression and Purification of gbr22

__ Introduction: __ Protein expression is the processes of over expressing a target protein by using a cloning (expression) vector. The expression of a protein is done on a basic E. coli host strain from a variety vectors with different tags and/or fusion partners.(1) The processes of protein purification involves the target protein being filtered out of the culture medium. This is done by utilizing several techniques. In this lab the technique utilize was filtration by affinity column in which a 6-HIS tag is added to the target protein. The production of specific proteins was once the domain of experts, but the development of simple, commercially available systems has made the technology more widespread.(2) This has resulted in a more cost effective way of producing the target proteins, this has significantly aid research since its first implementations. In addition with a processes of better analyzing the results obtained from the protein expression and purification this has led to more younger researchers to be enable to use this techniques.

__ Materials & Methods: __

In the first part of the labs the protein expressing E.Coli were prepared to express the gbr22 purple protein. This was done using a heat shock treatment followed by an ice shock treatment. This allowed the plasmid to be introduced into the inside of the E.Coli bacteria in order to be able to over express the gbr22. Then the E.Coli were cultured in a petri dish. Following that the bacteria were cultured in an LB media filled Erlenmeyer flask. Then the E.Coli bacteria were lysed and there DNA and RNA were broken down. Followed by a centrifuge to eliminate all the unwanted proteins that were in the solution. The purification was done using a affinity column, but in order to be able to use it the protein prior to the expression had a six-HIS tag sequence added to the C-terminus end. Using a Ni-NTA to capture the gbr22 from falling down the column while letting the unwanted proteins flow into the wash 1 and wash2. Then a high concentration of imidazole was use to free the gbr22 protein from the Ni-NTA. The result was analyzed using nanodrop to figure out the concentration of protein obtained and a protein gel to show the purity of the protein collected in the experiment.

__ Results: __

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__ Discussion: __ Lysozyme is used to break up the cell wall bonds, this inorder to prepare the protein for extraction. While Benoznase was utilize to break up the DNA and RNA strands. This helps in the purification of the protein to allow the DNA and RNA to be removed from the solution. In nature its extremely rare to find a protein with 6 HIS in a row, this is why the 6-HIS tag is so efficient in purification by affinity. The 6-HIS tag works by attaching to a Ni-NTA which binds really well to the shape of the HIS. Though the protein can be release using high concentration of imidazole. This since imidazole has a very similar shape to Histamine. In Sample 1, contain all the plasmid proteins no this was taken right after the lysozyme was added to break up the cell wall. Sample 2, contained the plasmid with the proteins and the DNA and RNA broken up this was why the band was more smooth. Sample 3, was taken from the wash 1 this should have contain everything that wasn't pbr22 since the pbr22 was stuck to the Ni-NTA because of the 6-HIS tag. Sample 4 is the wash 2, which should contain everything remanding after wash 1, but pgb22 since the pgb22 should have stayed in the column chromatography. Sample 5 is the elution 1 which should contain most of the pgb22 but to ensure that non was left in the column after elution 1 a second elution is done which is sample 6. The Wash buffer had a low concentration of imidazole while the Elution buffer had an extreme high concentration of imidazole in order to release the 6-HIS tag of the protein.  Using the gel results it was determine that no other protein band was found in conjunction to the pgb-22 this made the estimate of the purity to a 95+% pure.

__ Conclusions: __

In the three step lab a protein was over expresses using an expression E.Coli bacteria. Followed by a protein purification process and a series of tests to analyze the concentration and purity of the protein collected. This process was an effective way of mass producing proteins of intrest and as long as the purification process is maintained sterile This process can be use in future labs to obtain pure forms of the protein of interest. Though there is an additional step that was not taken in the purification process done in this labs that is crucial in obtaining a high purification yelled. Once obtaining that pure form of the target protein it can be used to test out the results of Virtual Screening by being able to analyze the effects of the legends obtained as top hit to the target active site of intrest. There are some sources of error since it still unsure of the plasmid used was not contaminated from the beginning. Other variables are the wrong measurements of concentrations and preparation of the solutions used though the labs.

__ References: __

1. Scholz, J., Besir, H., Strasser, C. & Suppmann, S. BMC Biotechnol. 2013 Feb 14;13:12. doi: 10.1186/1472-6750-13-12. 2. Nat Methods. 2008 February; 5(2): 135–146.