GAUTAM+W

__ **Title**: __** Expression, Purification, and Characterization of Protein (gbr-22) from E. Coli **

Specialized proteins that are of interest are produced and isolated for a variety of reasons including manufacturing for biotechnology, industry, or even for drug discovery purposes [1]. A process of steps must be taken to manufacture proteins for specialized purposes: protein expression, purification, and characterization. Protein expression involves inserting plasmid DNA, which encodes the sequence of the particular protein being expressed, into a host cell via heat shock or electroporation. E. Coli is a common host cell due to its low cost, high growth rate, high expression levels, and simplicity. The two main methods of transformation are electroporation and heat shock, heat shock being lower if efficiency but cheaper and easier [2]. Heat shock involves subjecting the host cell to higher temperatures such that the cell line is derived enough to contain small pores of which the plasmid DNA can enter through. After expression, the protein must be expressed by the host cell and then purified once enough proteins are present. Purification involves the addition of N-terminal Hexahistidine to the gene sequence of the protein. This allows for the simple process of immobilized metal affinity chromatography (IMAC) to be used to purify the protein while not affecting the characteristics of the protein [3]. In this experiment, E. Coli bacteria was used as the host cell to express the recombinant DNA (plasmid), pGEM-gbr-22, a purple fluorescent protein found in Corals from the Great Barrier Reef region. The gbr-22 protein was purified due to the addition of the N-terminal Hexahistidine and the concentration and purity was measured through characterization. The gbr-22 protein will be expressed in E. Coli, purified through IMAC, and the characterization will reveal a substantial amount of protein that will be 100% purified and isolated with no other proteins remaining in the solution.
 * __ Introduction: __**

Protein Expression E. Coli bacteria was added into 2 transformation tubes (25uL). 1-2uL of plasmid DNA, which contains gbr-22 protein, was added to the experimental transformation tube. Both tubes were heat shocked in a water bath for 45 seconds. Ampicillin was added to both plates and starter cultures were grown. A sample of E. Coli that expressed the gbr-22 protein was placed into a flask along with LB broth and Ampicillin, and a large culture was grown overnight in the shaking incubator. After the purple protein was over-expressed (solution turned purple), the solution was centrifuged and the solution was disposed so that only the pellet remained, which contained the E. Coli bacteria that have gbr-22 protein expressed. Aseptic technique was used when handling bacteria, and solutions were all placed under ice when not in use.
 * __ Materials & Methods: __**

Protein Purification Cyanase and lysozyme was added to the lysate and it was centrifuged for 20 minutes at 14,000 rpm. The lysate was further purified through syringe filtering. Ni-NTA resin/buffer mix was added to the lysate and the solution was run through a column filter, the flow through was collected. Wash buffer was run through and the ‘waste’ was collected. Elution buffer was run through the column twice, and the solution that flowed through was collected both times separately. Nanodrop sprectrophotometer was used to measure the concentration of gbr-22 protein in the elution 1 collected at wavelengths 280nm and 574nm. The yield of gbr-22 protein was calculated.

Protein Characterization Samples that were taken throughout each step in protein purification and expression were heated and centrifuged after adding a purple dye (loading buffer) to each sample. The electrophoresis module was set up and each sample was placed into the wells of the gel. A MW standard (Page Ruler Pre-stain Ladder by Thermo Scientific, # 26616) was added to the first well to base the sizes of the protein’s in the samples. A 200V electric current was run through the gel (for 25 minutes) which allowed the protein to run down the gel, proteins of different sizes stopped at different heights. The gel was stained by mixing in imperial protein stain and destained overnight. The gel was dried at 75 degrees Celsius on Gradient cycle for 1.5 hours and analyzed.


 * __ Results: __**















The weight of the pellet from protein expression (figure 5) was .36g. In protein purification, the maximal absorbance wavelength of gbr22 was at 574nm. The yield of the protein was calculated to be .099g/L at 280nm and .05g/L at 574nm, calculated using Beer's Law A=Ebc. In protein characterization lab, the estimated size of the protein, according to the dried gel is about 26 kDa. The purity of the elution 1 solution is about 40%. A=Ebc [At 280nm] .15=(38880)(1)(C) C=3.84E-6 mol/L * MW (257940.2g) = 0.99 g/L.

A=Ebc [At 574nm] .23=(118300)(1)(C) C=1.94E-6* MW (257940.2g) = 0.5 g/L. In the protein expression cultures, no bacteria was grown on the control plate since ampicillin was added to the plate which is an antibiotic that kills bacteria. However, the experimental E. Coli were able to grow even though Ampicillin was added because the gbr-22 plasmid also contained genetic information that provided resistance to the bacteria which incorporated the plasmid DNA with their own DNA. The bacteria that didn’t incorporate the plasmid died and the ones survived must have had to incorporate the plasmid DNA, indicating that they also incorporated the gene for producing purple protein. The pellet collected after centrifuging is a collection of all the bacteria which expressed the protein, while the waste included LB broth and ampicillin which were no longer needed. The cells were lysed using lysozymes to breakdown the cell wall of the bacteria to release the protein which is found in the cytoplasm of the bacteria. Cyanase was added to the solution in protein purification to digest the bacterial DNA. Centrifuging and syringe filtering filtered out broken down cell wall and other cellular debris, leaving only bacterial proteins in the solution. A Ni/Resin buffer was added to a column and the lysate was poured into a column filter. The Ni binds to the histidines at the outer surface of proteins (if they have any) which make them too big to flow through the column. Gbr-22 protein has 6 histidine tags, thus the flow through was waste. There are some proteins that have 1-2 histidines that the resin can bind to, thus a weak wash buffer (low concentration of imidazole) was used to filter out these proteins. Imidazole breaks the bond of Ni to histidine which allows those proteins to flow through. A low concentration of imidazole will break the bonds of Ni to proteins with 1-2 histidine tags. A high concentration of imidazole will break bonds of Ni to proteins with many histidine tags. Thus wash buffer contained a low concentration of imidazole to first get rid of proteins that the bacteria made which contained 1-2 histidine tags. Then elution buffer, which contains a high concentration of imidazole was used to let the gbr-22 protein flow through. In protein characterization there were many samples from different steps that were run in the gel. The first sample contained the cell fraction: which includes cellular debris and all proteins that the bacteria made. The next same contained the soluble fraction: includes the proteins that are found in the cytoplasm of the bacteria. Flow through sample contains proteins that don’t have any histidine tags, while wash sample contains proteins with weak histidine tags. Elution's 1 and 2 samples contain the gbr-22 protein along with 2 other proteins that have a lot of histidine tags as well. Elution 2 contains the remnants of proteins that didn't completely unbind from the Ni after addition of elution buffer the first time. According to the gel run the protein has a size of about 26 kDa, while measured to be 25794.2 g. The purity of elution 1 is about 40% since there are 3 proteins total but there seems to be a higher concentration of gbr-22 protein than the other 2. The Nanodrop was used to determine the quantity of the gbr-22 protein while characterization determined the quality or purity of the gbr22 protein. Some possible sources of errors include bad aseptic technique, incorrectly made wash/elution buffer, math errors, contamination during gel runs, and using incorrect Nanodrop techniques.
 * __ Discussion: __**

In the sequences of the lab series, first a purple flourescent protein (pGEM-gbr22) was transformed into E. Coli bacteria through heat shock and a culture was grown. The bacteria culture over-expressed the purple protein which was then centrifuged into a pellet. The pellet was lysed and further purification processes such as Ni-NTA affinity purification was used to purify the protein so that only the purple protein remained in solution. The concentration of the protein was determined via Nanodrop and different steps from both protein purification and expression were characterized via gel electrophoresis to determine the purity of elution 1. The concentration of protein was found to be 0.099 g/L and was found to be only 40% pure. Further steps must be taken to further purify the protein such that only gbr-22 remains. After the protein is completely purified, future applications can be accomplished for VDS such as testing the bindings of different ligands into the active site of the protein to determine which ligands are capable of inhibiting the protein and potentially be used as a drug.
 * __ Conclusions: __**

[1] Bradley, B.P.; Kalampanayil, B.; O’Neill, M.C. Protein Expression Profiling. //PubMed//. Ed. [Online] **2009**, 519, 455-468 [] (accessed April 16, 2013) [2] European Molecular Biology Laboratory: Protein Expression and Purification Core Facility. [] (accessed April 17, 2013) [3] Graslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B.M.; Bray, J.; et al. Protein Production and Purification. //Nat Methods//. Ed. [Online] **2008**, 5, 135-146 [] (accessed April 17, 2013)
 * __ References: __**