HYUN-YOUNG+L

__ Title: __ Protein Expression, Purification, and Characterization of gbr22 __ Introduction: __ Recently, the use of recombinant proteins in scientific research has increased greatly. With this, techniques and products for protein amplification and purification also developed greatly [1]. These techiques and science of protein expression, purification and characterization play a great role of wet lab of virtual screening. After computer puts out possible ligands for the protein, those ligands must be test in the wet lab to see if they really bind to the protein. The expression of protein usually involves a host organism or cell and genetically modified plasmids that is overexpressed in the cell. After the protein is expressed, it is then purified. The cell wall is broken down, DNA/RNA is digested. After centrifugation, only water soluble proteins are left. The protein of interest is extracted using various purification method. Chracterization of the protein shows the purity and expression of the protein. In this lab, the protein gbr22, a purple protein found in coral, was expressed, purified and characterized. If proper methods were executed, purple protein should be extracted and when the gel is ran, it should have one thick band indicating high concentration of one protein.

__ Materials & Methods: __ The first part of this lab was expression of protein of interest, which was gbr22. Plasmid pGEM-gbr22 (187.9 ng/µmL) was inserted into competent bacterial cells of E.coli BL21 (DE3) from NEB. The plasmid contained a gene for ampicillin resistance and the fluorescent protein that had six histidine residues appended at the C-terminus. After the protein was expressed in bacteria in the large culture, the cells were harvested using centrifugation. Next, the protein of interest was purified from the harvested cells. The cells were break opened using lysozyme. Also, Cyanase was added in order to dissolve DNA/RNA. Then the solution was centrifuged. This centrifugation isolated cell debris from water-soluble proteins. The supernatant liquid containing water-soluble proteins were purified using Ni-NTA affinity chromatography. The six histidine residues strongly bound to Ni2+ and wash buffer made with weak concentration of imidazole eliminated any other proteins that were loosely bound to the resin. The protein gbr22 was released from Ni resin by running elution buffer with strong concentration of imidazole which competed with the protein for Ni2+. Gel electrophoresis, SDS-PAGE, was used to separate the proteins in the collected samples. Total of six samples were taken along the above steps. Sample 1 was bacteria and liquid media right before harvesting. Sample 2 was taken after inserting Cyanase. Sample 3 was taken from first flow through of Ni-NTA affinity chromatography, Sample 4 from flow through of wash buffer, Sample 5 from first flow through of elution buffer, and Sample 6 from second flow through of elution buffer. Imperial protein stain was added to each of the sample and they were run through the gel electrophoresis along with prestained protein ladder from ThermoScientific (PageRulerTM, Product #26616, Lot#00117687, Exp: 02/14). After running the gel, the gel was stained and dried. __ Results: __ __ Discussion: __ Overall, the lab was successfully done. As seen in experimental and control plate, only those cells that incorporated plasmid survived. The purple color of the large culture and pellet shows that gbr22 was successfully expressed. After expression of the protein, it was purified. Before purification, the protein of interest was inside the E.coli cell. To break open the cell, lysozyme was added, which digested cell wall of E.coli (DE3). At this point, the protein was outside the cell, floating in the solution with other cell materials. To this solution, Cyanase was added, which digest DNA/RNA. After centrifugation, only water soluble proteins were in the solution. Digesting DNA/RNA was essential since they are too big to pass through Ni-NTA matrix.

The water soluble proteins were purified using Ni-NTA resin column. This system worked by negative Histidine tags of gbr22 attaching strongly to positive nickel ion. Because nickel ion is also bound to NTA, it is too big to pass through the column and able to hold on to gbr22. First, the solution was put in and let it flow through. The flow through of this step was called 'waste' and was put into well #4 in gel electrophoresis. This step eliminated all the proteins that did not have negative charge and therefore did not have gbr22 in it, which was indicated by its clear, colorless appearance. Next, a wash buffer of low concentration of imidazole was put into the column and let it flow through. This was called 'wash' and it eliminated all the proteins that are loosely bound to the resin matrix. The buffer was made using imidazole because it can replace HIS tag on Ni-NTA matrix. After wash buffer, the elution buffer was put it. This was a solution with very high concentration of imidazole which released gbr22. At this point, most of gbr22 was in the flow through of elution buffer and was indicated by its distinctive purple color. Another portion of elution buffer was put into capture rest of gbr22.

Elution 1 was used to determine the absorbance. Absorbance was measured at two different wavelengths, at 280nm and at 574nm. 280nm was the general maximum wavelength for any proteins because at 280nm, light is absorbed by aromatic amino acids. 574nm was the maximum wavelength of gbr22. The absorbance was 0.49 at 280nm and 0.07 at 574nm. Using the absorbance, concentration was calculated with Beer's Law. With the concentration, the final yield was calculated. At 280nm, the yield was 1.625mg and at 574nm, it was 0.765mg. Of the two calculated values, the yield at 574nm would be more accurate since concentration of only gbr22 was calculated.

After the protein was purifed, it was characterized using gel electrophoresis. Samples 1 through 6 was obtained from various steps along the expression and purification part of the lab. Sample 1 had whole bacterial cells before harvest, Sample 2 was after DNA/RNA was digested by Cyanase, Sample 3 was the waste flow through, Sample 4 was the wash buffer flow through, Sample 5 was the elution 1, with most of gbr22, and Sample 6 was elution 2, which captured rest of gbr22 that elution 1 did not. In the gel, prestatined protein ladder was put into Well1 and Sample 1 through 6 was put into Well 2 through 7. For Well 8,9, and 10, Sample 4,5, and 6 of partner's were put in. After loading the samples onto gel, the gel was run, which separated proteins by its molecular weight. The heavier proteins remained at the top of the gel while lighter proteins traveled to bottom of the gel.

As seen in Fig. 5, Well #6 and Well # 9 has two thick bands and one thin band. Each band represented a protein of a particular weight. Comparing with molecular weight of gbr22 and Fig. 6, the first band of Well#6 and 9 represented gbr22. The thickness and darkness of the band showed that expression was successful. Theoretically, Well#6 should have had only one band representing gbr22. The result showed that the purified protein solution had another high concentration of unknown protein and a low concentration of another unknown protein. One interesting aspect of the result is that Well#6 and Well#9 had same result although the samples were from two different solutions. This may indicate that the error in the result was not due to errors in expression of the protein. The plasmid could have had coding for those two proteins.

The hypothesis was that if proper methods were followed, gbr22 should be expressed, purified, and characterized. The result supported the hypothesis. Gel electrophoresis showed gbr22 was purified, but also indicated that purified solution had two other unknown proteins.

__ Conclusions: __ In this lab, pGEM-gbr22 was expressed in E.coli(DE3). The cell was broken open using lysozyme and DNA/RNA was digested using Cyanase. The solution containing gbr22 was then purified using Ni-NTA matrix. The collection of gbr22 was able to be observed due to its distinctive purple color. Using Nanodrop spectrophotometer, aborbance at 574nm, the max wavelength of gbr22, was measured. Using the concentration derived from the absorbance, the final yield was calculated to be 0.765mg. The purified protein was characterized using gel electrophoresis. The Elution 1 well had three bands total. This meant that there were three proteins in the sample. The result supported that the plasmid inserted into E.coli may had other genes for two unknown proteins. __ References: __ [1] Graslund, S.; Nordlund, P., Weigelt, J., et. al, Protein production and purification. Nat Methods //2008,// 5 (2), 135-46]][1] Graslund, S.; Nordlund, P., Weigelt, J., et. al, Protein production and purification. //Nat Methods// 2008, **//5//** **(2), 135-46**