Zamaria+J

** Introduction ** Proteins are one of the most essential macromolecules because they facilitate in biological functions so knowing facts about them gives a greater understanding of the world. Expressing, purifying, and characterizing a protein can discover unknown information but because no protein is the same, it can be done in a multitude of ways. In this lab, as in many others, the protein was grown in bacteria so that it could be studied without using human tissues. E. Coli is the typical recombinant host for studies because of its fast and inexpensive testing possibilities that allow analysis in relatively short periods of time [1]. Chromatography procedure allows the purity of a protein to be optimized through buffer suspension and controlling the recombinant protein to column size ratio [2]. Characterizing the purified protein allows judgment on if the purification was done correctly, if other molecular species are present, and estimations on the approximate protein concentration [1]. ** Materials and Methods ** The first part of the lab was to clone the gene and insert it into a DNA expression plasmid so that was done by working in a sterilized area and manner through placing 25 microliters of BL21(DE3) into a LB Agar Amp plate with plasmid DNA, putting it on ice for 30 minutes, and then heat shocking it in an incubator for 30 minutes at 37 Celsius. Colirollers were then used to spread the bacteria in the plate and left in a 37 Celsius incubator overnight to grow. The following morning a single colony from the multiple grown was chosen for two tubes and put with 5 ml of LB and put in the shaking incubator for 8 hours at 37 degrees Celsius and 200-350 rpm. 25 microliters of LB was then put into two Erlenmeyer flasks with ampicillin and 0.625 ml of the starting culture to grow for another 24 hours. Then the cells were harvested into a cell pellet by centriguation at 5000 rpm and 4 degrees Celsius for 10 minutes. The cells were then resuspended in a phosphate buffered saline by adding lysozyme and vortexing it. The second part of the experiment was lysing the E. coli cells by adding Benzonase then clarifying the lysate to keep sample for future reference. Buffers were then added and a syringe filter was used to get rid of the purple protein. Then transferring the resin and buffer into the conical tube did Ni-NTA affinity purification and a sample was taken. Two different buffers were then used to make elution samples. A Nanodrop spectrophotometer was used to estimate the concentration of the final purified protein at 280 nm and maximal wavelength. All of the samples from the past labs were put together with a 6x gel loading buffer and centrifuged then put on a heat block. Then using a mini-PROTEAN tank, a gel made, stained, and destained overnight. The next day, the gel was dried and from there it was compared to the standard to find out facts about the protein. ** Results ** Through the protein being expressed, purified, and characterized, the molecular weight of the protein, which was found by comparing the pre-cast gel to a standard molecular weight protein ladder was found to be 30 kDa. From this information, the estimate of the final protein, which was expressed as sample 5 in the pre-cast gel, was 70 grams.
 * Express, Purify, and Show Your Character **

















Beer’s Law (A=Ebc) calculations used during the lab

Determining the concentration of the protein in mg/ml at wavelength 280nm:

Absorbance = [(0.867+ 0.877)/2] = 0.872

Extinction Coefficient = 38850 g/mol

C=(0.872/38850)= 0.00002245 M

(0.00002245mol/L) x (1L/1000ml) x (38850g/mol) x (1000mg/1g) = 0.872 mg/ml

Determining the concentration of the protein in mg/ml at wavelength 574nm:

Absorbance = [(0.159+ 0.152)/2] = 0.1555

Extinction Coefficient = 118300 g/mol

C=(0.1555/118300)= 0.00000131M

(0.00000131mol/L) x (1L/1000ml) x (118300g/mol) x (1000mg/1g) = 0.1555 mg/ml Determining the yield Maximum wavelength:

280nm wavelength(0.872 mg/ml) x 5 ml=2.5mg

574nm wavelength(0.1555 mg/ml) x 5ml=6.875mg





** Discussion ** From this experiment, a lot of information was found but it was made off of assumptions. Because this was such a long experiment, error could have came at any time and caused the results to be off. The only actual problems that came about were during the expression part of the lab, which was trying to get the protein to grow and then selecting only one colony, and this was only a problem because of the tediousness of the task. The results are as accurate as possible. The samples used for the gel electrophoresis were all different parts of the experiment. Sample 1 had the gbr22 protein, soluble and insoluble; sample 2 had soluble part because Benzonase was used to remove the insoluble; sample 3 had all of proteins after it was ran through with PBS buffer; sample 4 was double the dilution of the previous because of filtration; sample 5 had the purified version of the gbr22 protein; and sample 6 had the rest of the proteins that remained after it was purified again. The “Wash” sample was made using 20 mM imidazole and the “Elution” samples were made using 250 mM imidazole, so the “Wash sample is more concentrated. The HIS tag system allows a specific protein to be easily separated from the other proteins in the cell by the histidine residues being attached to the C-terminus; without HIS tag, the fast and efficient methods of this lab would not have been possible. ** Conclusions ** The whole point of this lab is to overexpress Escherichia coli bacteria so that a protein that is encoded by a plasmid (pGEM-gbr22) of interest can be studied without using human tissues. The first lab was transforming the competent cells, growing the E. coli cultures to express the protein, harvesting the cells by centrifugation, suspending them in a buffer, and storing them at -20 degrees Celsius. The second lab was breaking open the E. coli cells that expressed the purple protein, clarifying it by centrifugation, purifying Ni-NTA affinity chromatography, and storing the purified version at 4 degrees Celsius. The last lab was to characterize the purified protein by using gel electrophoresis and UV-Vis spectroscopy to monitor the success of each separation step. From all of this, it was concluded that the fifth sample of the six taken was the best purified version of the protein and from it, the molecular weight was found to be 70 grams, which was estimated by the data that gave us 30 kDa from the standard ladder. The process done in this lab can be replicated to be a part of drug discovery by characterizing the proteins that GOLD gives as the best binding ligands. From now on, if molecular weight of a protein is unknown, a process has been learned that can allow that information to be discovered.

** References: ** [1] Nat Methods. **2008** Feb;5(2):135-46. Protein production and purification. [2] Encyclopædia Britannica Online. http://www.britannica.com/EBchecked/topic/227899/gel-chromatography. (accessed Apr 15, 2012).