Aldo+O

__ Title __
 * An Experiment with E. Coli: Protein Expression, Purification, and Characterization**

__ Introduction: __ The use of recombinant DNA has been a source of wide-spread research projects for the production of specific proteins using "hosts" that will ultimately serve as factories that produce the desired protein through DNA implantation and expression. Although there are several different methods to complete such processes, the most commonly used organism for the expression of proteins through recombinant DNA is the bacteria E. Coli. The process by which this is done is quite systematic, generally speaking, but it can get problematic as some factors including determining whether the protein should be expressed in bacteria, yeast, or in human cells, if the whole protein segment of the DNA should be expressed, or determining the best purification strategy can become quite difficult to overcome [1]. The overall process could be compressed as such: Obtain the proper cDNA that will be used, cloning that cDNA into an expression vector (usually an E. Coli one), use an RNA polymerase for the expression, place the organism obtaining the expression vector in a rich medium that promotes protein expression and allow the organism produce the desired protein, and, finally, purify the protein [2]. The main objective of this experiment was to successfully produce and purify the pGEM-gbr22 protein (found in Great Barrier Reef corral) which is purple in color (non-fluorescent) using the E. Coli BL21 (DE3) bacteria strain for the expression and production of this protein. Given that all procedures were conducted correctly, one can hypothesize that the final product of the purified protein will not only be composed of the desired protein - the contents, or products, of the protein expression will not be fully segregated from the pGEM-gbr22 protein and thus will not yield a 100 percent pGEM-gbr22 protein content.

__ Materials & Methods: __ In this experiment, the main materials needed included the bacteria E. Coli BL21 (DE3) strain, the plasmid containing the code for the pGEM-gbr22 protein, several test tubes, petri dishes, centrifuge tubes, pipette tips and pipettors, Erlenmeyer flasks, beakers, a spectrophotometer (Thermo Scientific, Wilmington, DE), a centrifuge machine, a gel electrophoresis device (mini-PROTEAN) and other materials commonly used for the proper expression, purification, and characterization of protein. The lab was divided into three parts. To begin, the recombinant protein was over-expressed in the E. Coli bacteria by placing 25 ul of competent bacteria into two transformation tubes (one control and the other with DNA. Soon after, the required plasmid DNA was added to the solution containing the bacteria (not the control tube) and went through a series of heat and cooling processes to promote implantation of the DNA. Petri dishes were acquired and 200 ul of SOC media were added to the tubes containing the bacteria. 50 ul of the bacteria/SOC mixture were added to two petri dishes, one labeled control and the other with DNA. The plates were stored in a 37 degrees celsius incubator overnight. Bacterial colonies were then swabbed from the plate and were placed into a tube containing a solution of LB broth and ampicillin. The bacteria was then allowed to grow on a shaking incubator for about a day in an Erlenmeyer flask containing the bacteria, LB broth, and ampicillin. A 500 ul sample (sample 1) of the purple solution containing the cells was obtained and stored. The cells were then harvested using a centrifuge machine, and 2.5 ml of 1x PBS solution was added to the tube and lysozyme was also added as well (to a concentration of 1mg/ml of lysozyme) with the harvested cells. Sample 2 was then obtained by adding 2 ul of benzonase to the tube and centrifuging it right after. The remaining liquid solution was syringe filtered. Wash and Elution buffers were then made by using 1x PBS and 20mM imidazole and 1x PBS PBS and 250mM imidazole solutions, respectively. Sample 3 was obtained after the protein was purified using column chromatography (.5 ml of Ni-NTA resin/buffer). Sample 4, 5, and 6 were obtained after the addition of 5 ml of wash buffer, 5 ml of elution buffer, and the remaining elution buffer. A Nanodrop spectrophotometer was then used to measure the absorbance of the Elution 1 sample at 280 nm and 574 nm. Samples 1-6 were then obtained and were placed in SDS-PAGE gel for the electrophoresis process. The gel ran for about 40 mins at 200V, and the MW standard used was the PageRuler by Fermentas. The gel was then acquired, stained for about 1 hour in a shaker, and was later dried at 75∘C for about 1.5 hours to complete the experiment.

__ Results: __

Beer's Law: A=Ebc So, Concentration (c)= A/Eb

For the protein measured at 280 nm: c=.79/((38850 1/M*cm)(1 cm) = .00002033 mol/L

For the protein measured at 574 nm: c= 1.34/((118300 1/M*cm)(1 cm)) = .0000113 mol/L

__ Discussion: __ At the end, most of the pure protein was obtained, but its purity was still questionable as the gel electrophoresis showed some light bands along with protein band in both sample 5' s. Although both samples were relatively pure, the second sample 5 seemed to have a smaller amount of bands in the Elution 1 lane, which correlates to a purer protein. The purity of the protein can be estimated to be between 50 - 75 % as there aren't any significant bands of the same color intensity as the purified protein band. Sources of error in the experiment could have came from improper measurement of the amounts of solution needed for each step, failing to work in a sterile environment, or faulty equipment. The molecular weight of the protein was estimated to be at about 25 kDa using the PageRuler Prestained Protein Ladder chart. Lysozyme was used in the Expression portion of this lab to breakdown the cell walls of the bacterial cells, which would then allow the exposure of the cell's proteins and other matter to be released into the solution for removal. Benzonase was used to decrease viscosity of the solution containing the cell's DNA and RNA byproducts. Benzonase essentially breaks down RNA and DNA, which have a relative high mass, and allows the solution to be more fluid for pipetting and other procedures. Sample 1 was pure bacterial cells obtained after they were harvested. Sample 2 was obtained after the lysate was added (the supernatant). Sample 3 came from the waste of the first column run. Sample 4 contains the wash/cell pellet solution. Sample 5 and 6 were both obtained from the elution buffer running through the column. The wash buffer was used to essentially 'wash' the pellet after the waste was removed. The elution buffer allowed the actual protein to be obtained into the collecting conical tube. The HIS tag system functioned as a binding affinity bridge for the protein to bind to the Ni-NTA added to the solution containing the protein. Using the Imidazole from the elution buffer, the protein was allowed to unbind from Ni-NTA and thus be collected into the Elution 1 and 2 containers for further examination.

__ Conclusions: __ Overall, the experiment went well and the main objective was achieved - to obtain, purify, and analyze the protein pGEM-gbr22 using recombinant DNA and the bacteria E. Coli to produce the protein. The results showed that by using correct methods, one can successfully obtain a desired protein using a host organism to produce the protein in high amounts with little to no effort by the researcher and, also, the molecular weight of the protein (ours was found to be approximately 25 kDa). Using the information/data gathered in this experiment, one can apply this knowledge to find unknown information of a protein target which includes molecular weight and absorbency using gel electrophoresis and a spectrophotometer, respectively. Also, enzyme assays could be created to further analyze/determine possible drug targets or inhibitory molecules.

__ References: __ [1] Graslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; Protein production and purification. //Nature Methods//. **2008**, 5(2): 135-46.

[2] Barbara, C.; Wells, J.; King, R.S.; The Purification, Cloning, and Expression of a Novel Luteinizing Hormone-induced Mitochondrial Protein in MA-10 Mouse Leydig Tumor Cells. //Journal of Biological Sciences.// **1994**, 269(45): 28314-28322.