KEVIN+E

__ Title: __ __ Growt, Isolation, and Determination of gbr22 __

Good job, Kevin. Mention the plasmid in the first sentence of your M&M

__ Introduction: __ Before, protein expression, purification, and characterization “was once the domain of experts, but the development of simple, commercially available systems has made the technology more widespread”1. This allows the overexpression of a recombinant protein to be practiced in many laboratories for education and research purposes. This three step process allows experimenters to manipulate and understand many things about particular proteins. Sometimes, proteins are in low concentration in organisms, however, through overexpression them become concentrated and easy to manipulate and purify. Rapidly growing bacteria containing the protein’s DNA produce large amounts for usage. Purification can lead to advances in enzyme inhibition assays and production of nonnative proteins for research purposes without invasive experiments. With the protein flowing free with broken up cell debris, affinity chromatography helps isolate the protein from all the other substances. Finally, using gel electrophoresis, characterization allows an analysis of the concentrations of proteins in various samples. Additionally, characterization allows that “oligomeric forms of the same (apparently pure) protein can be separated. This can be critical for protein crystallisation experiments.”2 In this particular lab, a plasmid pGEM-gbr22 will be used will be used for the three step process. It encoded for a purple protein found in the Great Barrier Reef. In all, if gbr22, the purple protein, from pGEM-gbr22 is overexpressed so that it can be worked with in large concentrations, purified removing insoluble cell debris from its expressed cell, and characterized for analysis of various protein concentrations, especially gbr22 elution, the recombinant protein analysis process will be complete.

__ Materials & Methods: __ For transformation, two 25µl centrifuged tubes of //Escherichia-coli// were heat shocked at 42˚C and 200µl SOC media added and shook in a 37˚C incubator for 30minutes. Mixture was plated on agar and stored in 37˚C incubator overnight. One colony, 10µl Ampicillin and 5mL LB was added to a tube and placed in incubator for 8 hours. 0.625mL of culture and 5mL LB was placed in 4˚C incubator for 1 day. 2.5mL 1xPBS and 50µl lysozyme were added to the pellet. For purification, 2.5µl cyanase was added to 2.5mL cell suspension. It was mixed, distributed, and centrifuged for 20minutes in 2.17 microcentrigure tube. Supernatant was transferred, leaving behind cell debris, and distributed into 14mL round-bottom tube with lysate through a syringe filter and 5mL syringe. Then .5mL Ni-NTA resin/buffer was added to the syringe on the ring clamp. The solution settled and ran into a waste beaker. Wash buffer was added and flushed the waste fluid out. 5mL of Elution buffer was added to collect gbr22 until fluid was no longer purple. This was repeated again. The nanodrop spectrophotometer read elution 1, gbr22, at 280nm and 574nm. In characterization, sample 1 was centrifuged and 200µl water, 40µl loading buffer was added to pellet. 10µl loading buffer was added to samples 2-6. The electrophoresis module was cleared with 2ml TGS and 20µl of each sample was run. Gel was shaken in plastic container with water trice. Imperial stain shook with protein for an hour, then washed with kimwipe overnight. The gel was covered with whatman filter paper and dried at 75˚C for 1.5 hours.

__ Results: __ Fig 1. Bacteria pGEM-gbr22 colonies (about 300) after one night of growth in 37 degree Celsius incubator. Fig 2. Fun plate bacteria after one night of growth in 37 degree Celsius incubator (saliva). Fig 3. Flask of protein (gbr22) and bacteria (pGEM-gbr22) cells after protein expressed and grown for one night in 37 degree Celsius incubator. Fig 4. Pellet of bacteria and protein gbr22 (.29g) after centrifuge and expression.

Fig 5. Final product of protein gbr22 after lysozyme added after expression process.

Fig 6. Elution 1 consisting of the gbr22 protein after filtration of elution buffer causing imidazole and nickel to stick and remain in the column while protein flowed into the tube. Fig 7. Elution 2 consisting of remaining gbr22 protein from filtration process with imidazole and nickel. Fig 8. Nanodrop screen shot of elution 1, gbr22, at 280nm with a yield of 1.13 mg. Beer's Law calculation: A=ebc .27=(118300 L/(mol*cm))*(1cm)*c c= 2.28x10^-6M molecular weight of gbr22= 25794.2g/mol (2.28x10^-6 mol/L) x (25794.2g/mol) x (1L/1000mL) x (1000mg/g) = .0589 mg/mL Therefore, the absorbance at 574nm does not match the absorbance at .23mg/mL. Fig 9. Dried gel of the protein gbr22 after characterization. gbr22 is slightly above 8th line of the MW comparison. MW= molecular weight standard, sample 1= cell fraction, sample 2= soluble fraction, sample 3= flow through, sample 4= wash, sample 5= elution 1, sample 6= elution 2. Fig 10. The ladder used in protein characterization, 4-20% Tris-Glycine SDS from PageRuler Prestained Protein Ladder. __ Discussion: __ Lysozyme is used to break apart the cell components. With the cell membrane broken down, the DNA is free inside of the solution, able to extract and manipulate. Cyanase reduces the viscosity of the solution by digesting DNA. This allows easier access to the protein itself, and not sticky substances along with it.

The HIS tag is inserted into the protein and attaches to it. Therefore, when Nickel is added to the solution, the protein binds to it, allowing all else in the solution to run through the filter. Then, when large concentrations of imidazole is added to the nickel and protein solution, the HIS tags are replaced by the binding of nickel and imidazole. The protein is then allowed to flow through and be collected.

Sample 1 is the cell fraction, the bacteria culture after incubation. Sample 2 is the soluble fraction of the bacteria and lysate, breaking down cell components. Sample 3 is the flow through after the lysate and resin and buffer are added to the column and flowed through material. Sample 4 is the wash. This is the waste after the wash buffer is added to Ni-NTA and the protein. Sample 5 is elution 1 which is the isolated protein after elution buffer is run through the column causing nickel to release the HIS tags on protein and bind to imidazole. Sample 6 is elution 2 which is the same and elution 1, just the second round of adding elution buffer to ensure all the protein is extracted.

The wash buffer contains a small concentration of imidazole aiming to remove the loosely bound proteins to resin along with other substances, leaving behind the strong resin-protein solution. In contrast, elution buffer contains a large concentration of imidazole in order to unbind HIS tags on the protein from nickel. The imidazole will bind to the nickel allowing the freed protein to run through for collection.

The known molecular weight of gbr22 is 25.7 kilo Daltons. Comparing to the ladder used in our gel, the protein in elution 1 is in fact gbr22, with the strong band slightly above the 25kDa line.

The elution 1 sample is about 90% pure, with a few trace bands around. This is not what was intended, but still presents the purpose in an attempt to isolate gbr22. The error in the experiment can come from many steps, but it is proven to be there since elution 1 is not pure of gbr22. Error in expression is overpopulating the bacteria so that the LB resources are not distributed enough. Error in purification is prevalent when the protein is transferred of filtered. Much depends on the correct concentrations added to the protein solution such as a low imidazole concentration may not collect all the protein. Error in characterization can come from the running of the gel and ensuring the lane is intact and it is run for a long enough time for separation of species.

__ Conclusions: __ During protein expression, gbr22 was added to Escherichia coli after heat shock and grown in culture. Eventually, it was isolated into a pellet for purification. In purification, the protein was extracted from the lysed cell solution. In a filter, the protein bound to nickel allowing impurities to flow through. Then, imidazole from elution buffer bound to nickel allowing gbr22 to flow through in isolation. During characterization, each sample from various parts of the lab was run in the gel, determining the components of each sample and how pure gbr22 was in elution 1. In the future, this can be used in VDS as a way to add isolate large amounts of proteins for experimentation and to determine the purity of these solutions for error analysis.

__ References: __ 1 Nat Methods. **2008 ** Feb;5(2):135-46. Protein production and purification. = = 2 European Molecular Biology Laboratory. Protein Expression and Purification Core Facility Protein Purification. [] (accessed April 15, 2013).