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__ Title: __ The process and analysis of protein production via purification and characterization.

Make intro more relevant to this lab

__ Introduction: __ Proteins have many wide ranging applications in the field of research, and thus efficient and cheap methods of protein production are of great interest to the research community. Included in these methods is using //E. coli// as a host to express the target protein because it is fast, and the results can be analyzed easily[1]. However, this method is useless unless the target protein can be isolated from the bacterial proteins for further use. Thus, ficient methods of protein purification have also been developed. One commonly used method is that of affinity chromatography where tags, commonly 6-HIS, are attached to the protein of interest and can be filtered out by using a binding matrix to which the tag will stick. Afterwards, the purified protein can be obtained by using an elution compound which will bind to the binding matrix causing the tags of the protein to unbind [2]. The intention of this experiment was to understand the process of bacterial protein production, isolate the produced protein, and analyze the purification process through protein characterization. It was hypothesized that the highest final yield after purification would result from the largest wet pellet obtained from the bacterial expression step.

__ Materials & Methods: __ 50 ul of competent bacterial cells, BL21(DE3) (New England BioLabs, Ipswich, MA) and 1.597 ul of plasmid pGEM-gbr22 were heat shocked at 42°C for 45 seconds. (Mention Control and Experimental plates)  200 ul of SOC were added. After 30 minutes in the shaking incubator, the solution was distributed onto an ampicilin positive agar plate which incubated for 24 hours. A starter culture was made from one of the growing colonies, 5 ml of LB, and 10 ul of ampicilin. After 8 hours, a large culture was made from 0.625 ml of the starter culture, 50.1 ul of ampicilin, and 25 ml of LB. 50 ml of the culture was centrifuged (What type of centrifuge) . Only the pellet was mixed with 25 ml of 1x PBS and 51.02 ul of 50 ug/ml lysozyme and stored in a the freezer for purification.

 The stored tube was incubated at room temperature for 20 minutes and then for another 15 minutes after adding 2 ul of Cyanase. The lysate was centrifuged and the supernatant was run through a 0.22 um syringe filter (What brand of filter). The remaining solution was mixed with 0.6 ml Ni-NTA resin and flowed through a chromatography column several times: once with wash buffer (10 ml of 1x PBS with 20 mM imidazole) and twice more with the elution buffer (10 mL of 1x PBS with 250 mM imidazole). Samples were taken periodically for characterization. The Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) was used to measure absorbance readings of the two elution samples at 280 nm and maximal wavelength.

 The protein purification process was then analyzed through characterization. Sample 1 was centrifuged and its pellet was mixed with 200 ul of water and 40ul of loading buffer. Samples 2-6 were mixed with 10 ul of loading buffer. These samples were loaded into a gel (What type of gel) along with a prestained molecular weight standard (Thermoscientific, Waltham, MA). After the gel was done running, it was stained with Imperial protein stain. It was then destained overnight and dried at 75°C for 1.5 hours.

__ Results: __ Show second trial as well The concentration of Elution 1 was found was found to be 5.58E-2 mg/ml or 2.16E-6 M (0.084/(38850 L/mol cm *1 cm)) at 280 nm and 2.51E-2 mg/ml or 9.72E-7 M (0.0115/(118300 L/mol cm * 1cm)) at maximal wavelength (574 nm) by using Beer's Law (A=Ebc). These concentrations resulted in final yields of 2.79E10-1 and 1.26E-1 for 280 nm and maximal wavelength, respectively. Show how yield is calculated from volume and how molecular weight is used to convert to mg/mL



__ Discussion: __ Colonies growing on the ampicilin positive plate (figure 1b) indicated that the protein (pGEM-br22) was successfully taken up because the plasmid also contained an ampicilin resistant gene. After centrifuging, the wet pellet remaining weighed 0.40 grams and contained the bacterial cells transformed with the purple plasmid as can be seen in figure 3.

During the purification process, lysozyme digested the bacterial cell wall and allowed the protein to float freely in the solution, while Cyanase digested the DNA and RNA in the mixture, leaving mostly protein in the solution. The remaining solution, however, still contained protein that were not the purple protein of interest. Thus, by running the solution through the chromatography column, the protein was purified by using the Ni-NTA buffer/resin which bound with high affinity to the HIS tags found on the purple protein. A wash buffer containing a low concentration of imidazole was run through the protein and Ni-NTA buffer/resin solution in order to cause loosely bound protein to come off of the beads of the Ni-NTA. The imidazole can also bind to the nickel in the beads and act as a competitor for the HIS tags. Ideally, at low concentrations, all of the protein that does not bind as tightly as the purple protein with six HIS tags will filter out, but as can be seen in Figure 7, protein characterization indicated that the purity of the sample was only approximately 70%. The elution buffer contained high concentrations of imidazole causing the purple protein to unbind from the beads and filter out separately afterwards.

Sample 1 was collected during bacterial expression when the protein was still located inside of the bacterial cell and thus has the most bands. Sample 2 was collected from the supernatant after the cell was lysed and contained a mixture of (soluble) bacterial proteins and the purple protein and has fewer bands than sample 1. Sample 3 was collected after letting the protein and Ni-NTA resin flow through the column and contained proteins that could not stick to the nickel causing the band pattern to be similar to sample 2 but lighter for the purple protein band. Sample 4 was collected after the wash solution was run through and contained proteins that were loosely bound to the Ni-NTA resin. Sample 5 was collected after the elution buffer was run through once and contained the purple protein and thus had one dark band indicating the purple protein. Sample 6 was collected after the elution buffer was run through a second time and also contained the purple protein at lesser concentration causing a lighter band at the same size marker. When comparing the band to the molecular weight standard, the band corresponded to approximately 27 kDa. This could not be compared to a molecular weight found in the purification process because the spectrophotometer only indicated concentration of the elutions. <span style="color: #ff0000; font-family: Arial,Helvetica,sans-serif; line-height: 1.5;">(MW found during the purification process was a theoretical value calculated from the primary sequence so technically you could compare it)

<span style="font-family: Arial,Helvetica,sans-serif;">Possible errors were in the purification process were not having a high enough concentration of imidazole in the wash buffer to purify the solution 100% and possibly not having enough lysate so that the protein actually obtained were at low concentrations. This caused problems in analyzing the solution with the Nanopure spectrophotometer because the concentrations were so low that it was hard for the spectrophotometer to pick up a signal resulting in a poor curve (figure 6). An error that occurred in the characterization process was not filling both chambers with buffer solution causing the gels to run very slowly. The gels were taken out before they were done running and thus have bad resolution. It is unclear as to why the drying process produced so many cracks in the gel.

__ Conclusions: __ In this lab, bacterial cells BL21(DE3) were transformed with the plasmid (pGEM-br22), which encodes for a gene expressing purple protein. These bacterial cells were incubated and suspended in log phase growth where many of them were expressing large amounts of the purple protein. The bacterial cells were lysed open to obtain the protein inside of them, and the protein solution was filtered using a syringe filter and purified using column chromatography. The samples from the purification process were then run using gel electrophoresis to analyze how efficient the purifcation process was. It was found that bacteria can be used to produce large amounts of target protein only if the purification of the protein is successful, which it was not in this experiment. These three processes have important applications where they can be used to efficiently produce protein via a bacterial vector and purify the target protein which can be used as a marker or as a potential drug target.

__ References: __ 1. Acton, T. B.; Albeck, S.; Almo, S. C.; Anderson, S.; Arrowsmith, C.; Atwell, S., Protein production and purification. // Nature Methods //** 2008, ** // 5 // ,(2), 135-146. 2. European Molecular Biology Laboratory. Protein Expression and Purification Core Facility. [] (accessed Apr 15, 2013).