KrishnaP

Krishna Patel 4/18/11

**Bacterial Protein Expression, Purification, and Characterization**

//__Objective/Introduction:__//  Biotechnology has greatly progressed in the last few decades especially in the field of molecular genetics. Technology has been used for protein expression to obtain amplified quantities of proteins that were once previously difficult to acquire. //E. coli// is often the bacteria of preference as it is harmless to adults. Bacteria are used as expression hosts by transforming the protein of interest in a plasmid vector for the bacteria to incorporate in their own DNA. Bacteria are used for gene expression to amplify the protein of interest [1]. There are several purification strategies to isolate the protein. One such strategy is chromatography in which the solution containing the protein flows through a column packed with various materials. Different proteins interact differently with the column material, and can thus be separated to elute the protein from the column. Other protein purification methods include ultracentrifugation and extraction. Expanding technology has clearly led to numerous methods to allow for protein production and purification [2].

In these labs, the purpose to overexpress a recombinant protein (gbr22 purple protein) in bacteria, purify the protein, and use a gel electrophoresis to analyze the protein samples. The importance of these labs is that it builds a foundation to amplify and purify proteins that could be used as potential drug targets in the Virtual Drug Screening research stream.

//__Materials and Methods:__//  Several safety precautions were taken during these labs. Gloves, goggles, and lab coats were used throughout because toxic chemicals were used. Also, all biohazardous waste was treated and disposed of properly.

In the first part of this lab, the first step was to transform //E. coli// bacterial cells with gbr22 protein. 3 plates were used including, one experimental plate with DNA, one Control plate that has no DNA, and one plate for ‘fun’ with bacteria from coughing on the plate. The SOC mixture that either contained the bacteria or did not was accordingly added to the appropriate plate. The starter culture was then grown using LB that was supplemented with 100 m g/mL ampicillin. The starter cultures were transferred to Erlenmeyer flasks and set in the incubator to grow over night. After the cultures were purple, the cells were harvested. Next the cells were resuspended in phosphate buffered saline by adding lysozyme to a final concentration of 1 mg/ml.

In the second portion of the lab, the protein was purified. First the //E. coli// were lysed with the lysozyme added in the first part of the lab. The lysate was then clarified and labeled as sample 2. The lysate was then syringe filtered to discard large particulate matter. The protein bounded to the resin using Ni-NTA resin/buffer mix. The resin and buffer were then transferred to column chromatography and the resulting sample was labeled as sample 3. The Ni-NTA was then washed through the column with the wash buffer and the resulting sample was labeled as sample 4. The gbr22 protein was released from the Ni-NTA by running the column with the elution buffer. The resulting sample was labeled as Elution 1 and the process was run again to obtain Elution 2. The Nanodrop spectrophotometer was used to estimate the concentration of the final purified protein.

In the last portion of the lab, the protein was characterized. The SDS-page gel samples were prepared and assembled in the electrophoresis module. The wells were loaded using a needle and syringe to clear out the wells and a micropipette to add the samples. Next, the gel was stained by adding imperial protein stain. After a short waiting period, the gels were destained and placed in the orbital shaker overnight. The gel was placed on Whatman filter and Saran Wrap. Lastly, the gel was dried in a vacuum in the Biotech lab.

//__Results:__//     <span style="font-family: 'Times New Roman',Times,serif;"> <span style="font-family: 'Times New Roman',Times,serif;"> <span style="font-family: 'Times New Roman',Times,serif;"> <span style="font-family: 'Times New Roman',Times,serif;"> <span style="font-family: 'Times New Roman',Times,serif;"> <span style="font-family: 'Times New Roman',Times,serif;"> <span style="font-family: 'Times New Roman',Times,serif;"> <span style="font-family: 'Times New Roman',Times,serif;"> <span style="font-family: 'Times New Roman',Times,serif;"> <span style="font-family: 'Times New Roman',Times,serif;">

<span style="font-family: 'Times New Roman',Times,serif;"> Sample 1 (Well 3 and 10): Cell Lysate <span style="font-family: 'Times New Roman',Times,serif;"> Sample 2 (Well 4): After Centrifugation <span style="font-family: 'Times New Roman',Times,serif;"> Sample 3 (Well 5): Flow Through <span style="font-family: 'Times New Roman',Times,serif;"> Sample 4 (Well 6): Wash <span style="font-family: 'Times New Roman',Times,serif;"> Sample 5 (Well 7): Elution 1 <span style="font-family: 'Times New Roman',Times,serif;"> Sample 6 (Well 8): Elution2 <span style="font-family: 'Times New Roman',Times,serif;"> Protein Ladder (Well 2 and 9)

<span style="font-family: 'Times New Roman',Times,serif; line-height: 0px; overflow: hidden;">

<span style="font-family: 'Times New Roman',Times,serif;">Figure 1 shows the three plates from part 1 of the lab including the control plate, experimental plate, and fun plate. No bacteria grew on the control plate as intend. Bacteria colonies are present in the experimental plate but they are not seen as purple because they were not expressing the purple protein at the time. The fun plate failed to grow any bacteria on it. Figure 2 shows the two cultures of purple bacteria that grew in the incubator overnight. Figure 3 shows the cell pellet after centrifugation. Figure 3A shows a protein pellet that weighs 0.36 g and Figure 3B shows a protein pellet that weighs 0.24 g. Figure 4 shows Elution 1 and 2 acquired after protein purification. Figure 5 and 6 shows the absorbance spectrum at 280 and 574 nm for the protein. At 280 nm the yield for the average of the trials was 0.662 mg. The yield for the maximal wavelength (574 nm) was 0.201 mg.

<span style="font-family: 'Times New Roman',Times,serif;"> Beers Law: A = εlc

<span style="font-family: 'Times New Roman',Times,serif;">**Yield for 280 nm Wavelength** <span style="font-family: 'Times New Roman',Times,serif;"> A = Average absorbance at 280 nm = (0.22 + 0.225) / 2 = 0.2225 <span style="font-family: 'Times New Roman',Times,serif;"> ε = Extinction Coefficient = 39100 M^-1 cm^-1 <span style="font-family: 'Times New Roman',Times,serif;"> l = Path Length = 1 cm <span style="font-family: 'Times New Roman',Times,serif;"> Molecular Weight = 25794.2 g/mol

<span style="font-family: 'Times New Roman',Times,serif;"> 0.2225 = (39100 M6-1 cm^-1) (1 cm) (c) <span style="font-family: 'Times New Roman',Times,serif;"> c = (5.69 * 10^-6 mol/L) (25794.2 g/mol) (1 mg/mL) <span style="font-family: 'Times New Roman',Times,serif;"> c = 0.147 mg/mL

<span style="font-family: 'Times New Roman',Times,serif;"> Volume of protein in Elution #1 = 4.5 mL <span style="font-family: 'Times New Roman',Times,serif;"> Yield = (4.5 mL) (0.147 mg/mL) <span style="font-family: 'Times New Roman',Times,serif;">//Yield = **0.662 mg**//

<span style="font-family: 'Times New Roman',Times,serif;">**Yield for 574 nm Wavelength** <span style="font-family: 'Times New Roman',Times,serif;"> A = Average absorbance at 574 nm = (0.16 + 0.25) / 2 = 0.205 <span style="font-family: 'Times New Roman',Times,serif;"> ε = Extinction Coefficient = 118300 M^-1 cm^-1 <span style="font-family: 'Times New Roman',Times,serif;"> b = Path Length = 1 cm <span style="font-family: 'Times New Roman',Times,serif;"> Molecular Weight = 25794.2 g/mol

<span style="font-family: 'Times New Roman',Times,serif;"> 0.205 = (118300 M6-1 cm^-1) (1 cm) (c) <span style="font-family: 'Times New Roman',Times,serif;"> c = (1.73 * 10^-6 mol/L) (25794.2 g/mol) (1 mg/mL) <span style="font-family: 'Times New Roman',Times,serif;"> c = 0.0447 mg/mL

<span style="font-family: 'Times New Roman',Times,serif;"> Volume of protein in Elution #1 = 4.5 mL <span style="font-family: 'Times New Roman',Times,serif;"> Yield = (4.5 mL) (0.0447 mg/mL) <span style="font-family: 'Times New Roman',Times,serif;">//Yield = **0.201 mg**//

<span style="font-family: 'Times New Roman',Times,serif;"> Figure 7A show the gel before it is dried. Well 2 and 9 contain Fermentas Page Ruler Prestained Protein Ladder. Well 3 and 10 contain the cell lysate. Well 10 contains the cell lysate because the lysate did not enter well 3 cleanly. Well 4 contains the sample after centrifugation. Well 5 and 6 contain the flow through and wash respectively. Well 7 and 8 contain Elution #1 and Elution #2. Figure 7B shows the dried gel. The gel is broken into pieces because the vacuum was opened during the drying process before turning it off which caused a pressure change and broke the gel. Figure 8 shows the Molecular Weight standard of the Fermentas Page Ruler Prestained Protein Ladder.

<span style="font-family: 'Times New Roman',Times,serif;">//__Discussion:__//

<span style="font-family: 'Times New Roman',Times,serif;"> In the protein expression part of the lab, the results can simply be seen in Figures 1, 2, and 3. Figure 2 shows the cultures are purple, which means these bacteria did take up the purple protein and are expressing it. However, some sources of error did occur prior to this in the bacteria plates. The experimental plate grew bacteria colonies but they were not purple. Since colonies grew on the ampicillin resistant plate that means the DNA was transformed because the bacteria survived. However, a possible reason why the bacteria were not purple could be because the purple gene might not have been expressed at the time but was still part of the bacteria’s DNA. As a result, purple bacteria from another group’s plate was used instead to start the starter culture. The “fun” plate did not grow any bacteria because it was barely coughed on twice. Coughing directly did not place the bacteria on the plate and explains why nothing grew on this plate. The second portion of the lab can be summed up from Figure 4 as it shows Elution#1 and Elution #2. Elution#1 is slightly purple and Elution #2 is clear. The reason for this difference is because Elution #1 is intended to contain most of the purple protein while Elution #2 carries only the protein that failed to wash through in Elution #1.

<span style="font-family: 'Times New Roman',Times,serif;">The gel electrophoresis shows the results of this lab. The gel shows the six samples taken throughout the lab. One source of error is that during the drying process the gel broke. This is because the vacuum was opened while the drying process was still running and caused a pressure change to force the gel to break. In Figure 7, Sample 1 was the most dense as it contained the cell lysate. Well 3 and 10 contain the cell lysate and show the most bands because it contains the most proteins from all of the parts of the cell. One source of error occurred here when placing the cell lysate in Well 3 in which the sample did not properly enter the well because the cell lysate was too thick. Therefore, Well 10 was also used for the cell lysate to try and fix this error. Well 7 shows Elution #1 and a clear distinct band is present, which is the protein of interest. Since only one band exists under Well 7, it can be said that the purity of the final protein is 100%. Well 8 (Elution #2) has no distinct bands because very little protein was present in the elution as intended. Figure 8 shows the Molecular Weight standard of the Fermentas Page Ruler Prestained Protein Ladder. Using this figure it was determined that the approximate molecular weight of the protein in Well 7 (sample 5) is 25 kDa. Using an online convertor, it was found that 25kDa is equal to 4.1535 *10^-12 grams. This was multiplied by Avagadro’s number (6.022*10^23) to find the molecular weight of the protein which was 24999.4 g/mol. This molecular weight is rather close to the scientifically observed molecular weight of 25794.2 g/mol proving that the band in Well 5 is in fact our protein of interest. Thus, despite some sources of error, the gel electrophoresis was still successfully used to analyze the results and determine the molecular weight of the purified protein.

<span style="font-family: 'Times New Roman',Times,serif;">//__Conclusion:__//

<span style="font-family: 'Times New Roman',Times,serif;"> The lab illustrated how proteins can be isolated and amplified to later be used as potential drug targets. The objective was achieved as a recombinant protein, gbr22, was overexpressed using //E. coli//, the protein was purified, and a gel electrophoresis was completed to analyze the protein samples. The immediate next step for this lab would be to overexpress and purify other proteins that could be tested as a potential drug targets against various top 15 ligands found in GOLD.

<span style="font-family: 'Times New Roman',Times,serif;">//__Citations:__//

<span style="font-family: 'Times New Roman',Times,serif;"> 1) Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards, A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.; Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.; Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.; Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.; Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C.; Consortium, S. G.; Consortium, C. S. G.; Consortium, N. S. G., Protein production and purification. //Nat Methods// **2008,** //5// (2), 135-46

<span style="font-family: 'Times New Roman',Times,serif;"> 2) Protein Expression and Purification Core Facility. [] (accessed April 17, 2011)