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=** The expression, purification, and characterization of the protein fromp-GEM-gbr22 **=

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Recombinant proteins are widely used in biomedical sciences. They are obtained from the translation of cDNA in a bacterial species. Recombinant proteins have been used to answer several questions regarding when and where proteins should be expressed. In research, when scientists decide to target a certain bacteria, these proteins are utilized. Most research studies use Escherichia coli bacteria as its recombinant host for genes of these proteins. For the purpose of this lab, the plasmid p-GEM-gbr22 that encodes for fluorescent protein originally cloned from the Great Barrier Reef will be expressed by BL21(DE3), an E.coli strand. p-GEM-gbr22 carries a gene for ampicillin resistance; its purple protein will be expressed from E. coli cultures.=====

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A chromatography procedure will be carried out to purify the protein. With several resins, binding capacities, and binding strengths, the selectivity of the purification can be controlled by the concentration of imidazole in the elution buffers used during chromatography. To maximize purity, one should load the column with a slight excess over the predicted binding capacity [1]. The washing step is usually followed by an elution step that removes histidine rich proteins. ===== There are several methods for characterizing proteins. They include mass spectrometry, static or dynamic light scattering, and measuring protein thermal stability [1]. The most common technique used for characterization is the sodium dodecyl sulfate polyacrylamide gel electrophoresis. In this process, samples are introduced to an end of a porous gel and a power supply applies electric field across the gel. Since the distance of migration is related to the mass of the protein, the protein's mass can be estimated by comparison to molecular weight standards.

**Materials & Methods**
The lab was divided into three sections; protein expression, protein purification, and protein characterization. During the protein expression part of the lab, the bacteria (BL21-DE 3)’s protein was over-expressed by cloning the gene and inserting it in an expression plasmid. Three plates were made for the expression. One was the experimental plate with DNA in it. The other was a control plate without DNA, and finally, there was a fun plate that contained sneeze or swab from somewhere dirty. Then the bacteria were transformed with a new plasmid on the first day of the lab. Finally, on the second day of the lab a starter culture was grown using LB media and Ampicillin stock, the protein was expressed in a large culture and eventually harvested on the third day. The second section of the lab involved using buffers to lyse the e.coli cells. During the lab, wash buffers and elution buffers were made using PBS and imidazole. After binding the protein to resin using the resin/buffer mix, these buffers were used to elute and remove proteins that were loosely bound to the resin. The protein was purified using batch and column chromatography. At the end of the lab, samples from the chromatography using the resin/buffer mix, wash buffer (elution 1), and elution buffer (elution 2) were collected. During the third and final part of the lab, samples collected from the previous labs were used to create a gel in order to approximate the molecular weight of the protein. The previous samples that were stored in a fridge were put in a microcentrifuge to collect cell pellet then they underwent electrophoresis in the Mini-PROTEAN electrophoresis tank.

Bacterial Protein Expression Below are images from the bacterial protein expression lab that was conducted over several days. After transforming the competent bacterial cells, about 110 colonies was obtained. After haervesting the cells and centrifuging it, the resulting weight of the pellet was about 0.65g. Fig 1. Fun plate from the bacterial protein expression lab. The plate contains sneeze.
 * Results: **

Fig 2. Experimental plate with DNA in it. Colonies are represented by purple dots.

Fig 3. The control plate with no DNA. This plate has no colonies. Fig 4. Large culture used for overnight expression. The flask contains LB broth and ampicillin. The purple color is due to bacteria colonies.

Fig 5. Sample of a culture that has been centrifuged. The cells settle to the bottom to form purple pellets.

Protein purification Below are images from the protein purification lab. After washing and removing loosely bound proteins, it was dicovered that 5.7mL of elution 1 and 3.9mL of elution 2 soliutions were collected from the batch and column chromatography. During the Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) process, the concentration of the protein at the maximal wavelength was determined. Beer's law was used to manually disclose the yield. Absorbance at 280nm= 1.232 A=Ebc (1.232)= (33850) (1) c c= 3.64e-5 mol/L Using this concentration of protein, the yield (volume of purified protein) was discovered to be 0.0928512mg

Absorbance at 574nm= 0.1705 A=Ebc (0.1705)= (11830) (1)c c= 1.4e-6 mol/L Using this concentration of protein, the yield (volume of purified protein) was discovered to be 0.03611186mg Fig 6. Elution 1 and Elution 2 samples that was collected the batch and column chromatography. The elution 1 solution is purple and the elution 2 solution is clear. Fig 7. Spectra from the first reading of the protein solution at 280nm. The blue line shows the absorbance reading at exactly 280nm. Fig 8. Spectra from the second reading of the protein solution at 280nm. The blue line shows the absorbance reading at exactly 280nm. Fig 9. Spectra of the first reading of the protein solution at 574nm. The blue line shows the absorbance reading at exactly 574nm. Fig 10. Spectra of the second reading of the protein solution at 574nm. The blue line shows the absorbance reading at exactly 574nm.

Protein Characterization Below are images from the protein characterization lab that was conducted over several days. A prestained protein ladder from Fermantas was used in the gel. The gel consisted of 7 lane. The first lane was the protein ladder, the second lane was sample 1, the third lane was sample 2, the fourth lane was sample 3 and so on. A molecular weight standard obtained from the internet was used to approximate the molecular weight of the protein. From the molecular weight standard ( fig. 12), the molecular weight of the protein was estimated to be 25kDa. Fig 11. The wet gel before it was dried. Each column represents the samples. Fig 12. Prestained Protein Ladder. The ladder to the left represents the ladder that was used during the lab. From the ladder one can approximate the weight of the protein to be about 25kDa

**Di scussion **
At each stacge of the lab multiple data was collected and eventually used in future steps. During the protein expression part of the lab, cell pellets of about 0.65g was collected after the harvested culture was centrifuged. While purifying the protein and using the Nanodrop spectrophotometer, it was discovered that the maximum absorption wavelength where the protein absorbs is 574nm. At this wavelength the extinction coefficint turned out to be 118300. With the aide of meffCP-gbr22 protein sequence it was determined that the extinction coefficient at 280nm was 39100 and the molecular weight of gbr22 was 25794.2. With these information Beer's law was used to find the concentration of protein in the solution. When the 280nm wavelength was used a concentration of 3.64e-5mol/ L was obtained. When the 574nm wavelength was used a concentration of 1.4e-6mol/L was obtained. The concentration, and volume was used to find the yield of the protein. The yields turned out to be 0.0928512mg using the information from the 280nm wavelength and 0.03611186mg using information from the 574 nm wavelength. The yields were not the same but somewhat similar. Some mechanical or calculation error might be the source of the differences. After characterization, the molecular weight of the protein was estimated to be 25kDa. In the sample 5 lane, there were 4 bands present. If there was one extra band present the estimated purity should be about 12. Lysosyme is used to breakdown the cell wall of the bacteria then Cyanase is used to extract DNA from the bacteria. Sample 1 contains 50 ul of the protein culture that was harvested. Sample 2 contains 50 ul of the lysate from the Benzonase enzyme. Sample 3 contained 50 ul of the flow of waste solution collected from the Ni-NTA affinity purification. Sample 4 contains 50 ul flow of wash solution that was collected from the Ni-NTA affinity purification. Sample 5 contains 50ul flow of wash buffer from the Ni-NTA affinity purification and finally sample 6 contains flow of elution buffer from the Ni-NTA affinity purification. The purification process removed loosely bound protein in the HIS tag system.

** Error **
During the protein expression lab, the bacteria did not grow as expected so multiple steps had to be repeated until the bacteria grew. The number of colonies used was also increased in order to increase the chances of the bacterial growth. For other labs, errors could have possibly resulted from miscalculation and improper mixture or usage of concentrations and volumes for solutions.

** Conclusions **
The lab was a combination of three parts lab that consisted of expressing a bacterial protein, purifying it and letting it go through characterization via gel electropoiesis. The final goal of the lab was to deteremine the molecular weight of the protein. During the gel electropoiesis process, samples collected from previous labs were used in a gel whose protein ladder was compared to molecular weight standards. The entire process of this lab was long but was broken down into sections in order to relief the workload. These lab processes are often used by researchers in biomedicall sciences. They can continually be used in the future to determine molecular weights of recombinant proteins.

** References **
[1]. Nat Methods. **2008** Feb;5(2): 135-46. Protein production and purification. [2]. European Molecular Biology Laboratory. Protein Expression and Purification Core Facility. [] (accessed Apr 14, 2011).