KATHERINE+F

__ Title: __ Expression, Purification, and Characterization Techniques of Purple Protein Gbr22

Great job, Renee. Give captions titles: FIg 1., Fig 2., etc.

__ Introduction: __ //Escherichia coli // bacteria are often used to produce proteins necessary for biochemical research [1]. This process is called overexpression since proteins that are never produced or produced in low quantities in an organism can be manufactured for research methods such as enzyme inhibition assays. Molecular biology techniques such as PCR allow an organisms’ DNA to be manipulated to express nonnative proteins express [2]. For example, in this lab an //E. Coli// plasmid has been preemptively altered to express a fluorescent purple protein originally cloned from a coral from the Great Barrier Reef and ampicillin resistance. The resulting altered plasmid, pGEM-gbr22, was chosen for the experiment since it is easily followed due to its purple color. The gene for ampicillin resistance allowed for aseptic techniques to be used in the duration of the experiment because the transformed E. Coli were able to grow in ampicillin-containing environments (agar plates and LB). The fluorescent protein has six histidine residues attached at the C-terminus end of the protein. The hexa-histidine tag has an affinity for cations such as nickel [2].The underlying purification theory is based off of the fact that the nickel cations attached to gbr22 by the histidine tag can be arrested in a chromatography column [2]. The protein can be released from the Ni-NTA by adding imidazole, which out-competes the histidine residues for nickel at high concentrations.This purification process is accurate in theory; however, a validation method to verify the success of each purification step was taken [1]. Spectroscopy was used to measure the concentration of proteins in solution; however, it cannot usually distinguish different proteins in a mixture [1]. The most common technique used to learn the identity of proteins is SDS-PAGE gel electrophoresis [1]. In gel electrophoresis, protein samples are placed at one end of a porous gel and an electrical current is sent across the gel using a power supply. This causes the protein samples to cascade down the porous gel and stop at a certain point along the gel based on the size of the proteins in the sample.The purpose of this lab is to learn the theory behind and practice protein expression, purification, and characterization, all of which will be crucial in future Virtual Drug Screening research. The expected outcome was that the E. Coli BL21(DE3) bacteria will express the protein, causing them to be purple in color throughout the experiment, and gel electrophoresis would validate that gbr22 is the only protein present in the elution solution.

__ Materials & Methods: __ To express the purple protein, gbr22, competent //E. Coli// BL21(DE3) were transformed with the plasmid PGEM-gbr22 by heat shocking the bacteria in 42 degrees Celsius water for 45 seconds. The transformed //E. Coli// BL21(DE3)cells as well non-transformed //E. Coli// BL21(DE3) cells were plated onto agar plates containing ampicillin and left to incubate overnight. The following day a started culture and later a larger culture were grown in LB supplemented with ampicillin and left to incubate the in shaking incubator at 37 degrees Celsius and 200-250 rpm for 8-24 hours. The next day the cells were harvested and a 0.5 mL sample of the purple culture was centrifuged to find the pellet weight. The cells were then re-suspended in 2.5 ml of 1x phosphate buffered saline.To purify the purple protein, transformed bacterial cells were lysed using Cyanase and lysozyme, centrifuged for 20 minutes at 14,000 rpm, the supernatant was decanted using a micropipette, and then filtered with a 0.45 μm syringe filter. The solution was mixed with Ni-NTA resin/buffer mix and purified using a 20 ml Bio-Rad chromatography Econo column. The solution was washed with 5 ml of 20 mM imidazole and eventually the protein was eluted using 5 ml of 250 mM imidazole. Along the way various samples were collected for characterization use. The Nanodrop spectrophotometer was used to calculate the concentration of the protein at 250nm and 574 nm, the maximal wavelength. To characterize the protein the six samples collected throughout the last two labs were prepared using 6x glycerol gel loading buffer. Then, the SDS-PAGE gel was run using the mini-PROTEAN electrophoresis module and a Bio-Rad pre-cast gel for 25 minutes at 200 V. Then the gel was removed, stained with Imperial protein stain, and dried the next day using Whatman filter paper and a drying bed at 75 degrees Celsius on gradient cycle for 1.5 hours.

__ Results: __





















__Discussion:__ When taking into account the estimated purity of the sample, 75%, the total yield of the protein is approximately 1.85 mg. Lysozyme is an enzyme that metabolizes the components of cell walls; therefore, it was used in protein purification to break apart the cell and release gbr22 as well as other soluble proteins into solution. Cyanase was added to the solution to break down DNA and RNA of the lysed cells in order to reduce the viscosity of the solution. The hexa-histidine tag binds with affinity to cations such as nickel, and in our experiment nickel cations were attached to beads. The nickel beads keep the gbr22 protein retained in a chromatography column. Low concentrations of imidazole in the wash buffer discharge any extra proteins that may have low affinity for either the nickel or hisitidine. However, it takes high concentrations of imidazole such as in the elution buffer to outcompete the nickel for histidine. When this happens, the gbr22 is released from the column. The estimated purity of the elution is 75%, since there is not a band of equal intensity, but slight coloration on the electrophoresis gel for samples 5 and 6. There are several sources of error to address which explain why the purity of the sample is not the desired 100%. The purification process is meant to be accurate in theory, however if a protein within the column had an area of high histidine concentration, the nickel beads could have easily attached to them as well. These contaminating proteins then could have then eluted with the elution buffer along with gbr22. Also, if a contaminating protein was the same size as gbr22 it would not be differentiated from the gel electrophoresis.

__Conclusion:__ In this lab a purple protein gbr22 was over expressed in //E. Coli // cells, harvested, purified using a chromatography column, and characterized with a SDSPAGE electrophoresis gel. The key findings in this lab include the estimated purity of the final protein solution, 75%, and therefore the total yield of protein, 1.85 mg. Protein expression, purification, and characterization are essential techniques used in Virtual Drug Screening, and the general methods used for protein gbr22 are the most frequently used in biochemistry [1]. Overexpressed proteins are fundamental in order to complete enzyme inhibition assays, and in order to ensure accurate results the target protein must be purified and characterized.

__ References: __ 1. Structural Genomics Consortium, China Structural Genomics Consortium, Northeast Structural Genomics Consortium et al. Protein production and purification. //Nat Meth//. **2008**;5(2):135–146. 2. European Molecular Biology Laboratory. Protein Expression and Purification Core Facility. [] (accessed April, 16 2013).