MIN+S

__ Title: __ **Protein pGEM-gbr22 Expression, Purification, and Characterization**

__ Introduction: __ Many techniques have been developed to express and purify recombinant proteins to develop new drugs or manufacture enzymes [1]. BL21(DE3) is the most widely used //E. coli// strain for protein overexpression and often provides the highest yield of target protein relative to endogenous proteins. Thus for high-level protein production purposes, BL21(DE3) (New England Bio Labs, Ipswich, MA), is an appropriate //E. coli// strain [2]. By transforming bacterial cells with a DNA plasmid and growing a starter culture of bacteria, the over-expressed recombinant protein is able to be purified and characterized. The plasmid (pGEM-gbr22) is a purple fluorescent protein that can only be detected at 280 nm in a UV spectrophotometer. The expression plasmid, which is inserted into the BL21(DE3) bacteria, possesses a gene for Ampicillin resistance (Ampicillin is an antibiotic that inhibits peptidoglycan formation in bacteria) and has six histidine residues at its C-terminus. This hexa-histidine tag allows for fast and efficient purification of the protein because the histidine residues bind to cations (i.e. Nickel) which can be immobilized on a column then released from the Ni-NTA resin via Imidazole which has a high concentration and removes tightly bound proteins. In order to purify the overexpressed protein, the bacterial cells are opened with lysozyme to release the soluble proteins, the insoluble cell debris is removed by centrifugation, the lysate is clarified, and Ni-NTA affinity chromatography is used. During characterization, SDS, a anionic detergent denatures proteins and imparts a negative charge that is proportional to its mass is used; thus the distance of migration through the gel is related to the mass of the protein and the mass of the pGEM-gbr22 can be estimated by comparison to molecular weight standards. The purified protein is run on a gel by electrophoresis (samples from different purification steps are exposed to an electrical field across the gel) to estimate the purity and yield of the final protein product. UV-Vis spectroscopy measurements can be compared to determine concentration of the protein solution. It can be hypothesized that if pGEM-gbr22 is properly expressed in BL21 (DE3), purified with Ni-NTA affinity tag, and characterized through gel electrophoresis, it will yield high purity.

__ Materials & Methods: __ Reduce M&M to most critical details Twenty Five (just write 25) uL of competent bacterial cells BL21(DE3) (New England Bio Labs, Ipswich, MA) and 2 uL of DNA plasmid were mixed and placed on ice for 30 minutes to prevent denaturing. Then the tubes of solution were heat shocked in 43 degrees Celsius water bath for 45 seconds. 200 uL of SOC media was added to the mixture and was shaken in an incubator at 37 degrees Celsius for 30 minutes at ~250 rpm. Around 6 colirollers were placed on the 3 diferent plates (pGEM-gbr22 plasmid, no dna control, fun plate). 50 uL of bacteria/SOC mixture were pipetted onto each plate and the plate was rolled until bacteria was evenly spread. The plates were inverted and stored in a 37 degrees Celsius incubator overnight. The next morning, a single bacteria colony was transferred into 2 sterile culture tubes containing LB broth/Ampicillin media. The tubes were placed in a shaking incubator to grow (8 hours) at 37 degrees celsius and ~250 rpm. After incubation, 25 mL of fresh LB were transferred to a clean 125 mL erlenmeyer flask, ampicillin was added to make the final concentration 100 ug/ml and 0.625 mL of the starter culture was transferred into the flask and put back into the shaking incubator at 37 degrees celsius, ~250 rpm for nearly 24 hours. The following day, a purple color indicated that the media had grown enough and was ready to harvest. 500 uL of the cell culture were dispensed in an Eppendorf tube and centrifuged for 10 minutes at 5000 rpm and 4 degrees celsius in the Allegra X-15 Benchtop Centrifuge (Beckman Coulter, Inc., Brea, CA). The purple pellet at the bottom of the tubes were saved and the liquid was decanted into a waste receptacle. The pellet was resuspended 2.5 mL of 1xPBS with 50 ug/mL of lysozyme for a final concentration of 1 mg/mL. The tube was vortexed one last time and stored in the -20 degrees celsius freezer.
 * Protein Expression**

The conical tube containing 2.5 mL of pellet cells was thawed in a beaker of water to ensure complete lysis. Then once the mixture turned viscous, 2 uL of cyanase was added (added benzonase) to the 50 mL conical and incubated at 15 minutes in room temperature. The lysate was distributed into two 1.7 ml microcentrifuge tubes and centrifuged for 20 minutes at 14,000 rpm at 4 degrees celsius. 50 uL of the supernatant was saved as Sample 2. Using a P1000, the liquid supernatant was transferred to a clean 15 ml conical tube then filtered through a 0.45 uL syringe and 0.5 mL of Ni-NTA resin was added to bind the protein. The mixture was filtered through a 20 mL Bio-Rad chromatography Econo column and 50 uL of the flowthrough sample was obtained (Sample 3). The Ni-NTA resin was washed with 5 mL of 20 mM imidazole in 1xPBS solution by adding it to the top of the column and letting it flow into a different 10 ml round bottom tube (Sample 4). Then 5 mL of 250 mM Imidazole in 1xPBS was dispensed into the column and was collected (Elution 1 as Sample 5). This was repeated by adding 5 mL of elution buffer and collecting in a conical tube (Elution 2 as Sample 6). The striping procedure was followed to preserve Ni-NTA and the Nanodrop Spectrophotometer (Thermo Scientific, Wilmington, DE) was used to measure the concentration of the final purified protein at 280 nm.
 * Protein Purification**

Samples 1-6 collect during the protein expression and protein purification labs were prepped for gel electrophoresis. Sample 1 was prepared by pipetting 500 uL of water into a 1.7 mL microcentrifuge tube and centrifuged for 5 minutes at 5,000 rpm. The cell pellet was retained and resuspended in 200 uL of nanopure water and 40 uL of loading buffer. For Samples 2-6, 50 uL of the 10 uL 6x loading buffer was added to each and heat shocked at 95 degrees celsius for 5 minutes then centrifuged at 5,000 rpm at 2 minutes. The electrophoresis module was assembled with the pre-cast gel in the mini-PROTEAN tank with 500 ml 1x TGS. Each lane was cleared using a ~20 gauge needle and syringe with a milliliter of TGS buffer injected into the well. Then 7 uL of the MW standards, 20 uL of the six samples + (Samples 4-6) was loaded into each of the wells using a P20 micropipetter. The voltage was set to 200V and run for 25 minutes. The gel cassette was removed and rinsed in nanopure water 3 times, stained with Imperial Protein Stain for 1.5 hours, and rinsed three times again with nanopure water. To destain the gel, nanopure water was added to cover the gel (~100 mL) and a kimwipe was placed in the dish to soak up excess stain. The gel was washed overnight on the orbital shaker to remove background staining. The following dat, the gel was placed on Whatman filter paper and covered with Saran Wrap then laid on the drying bed at 75 degrees celsius for 1.5 hours. The dried gel was analyzed.
 * Protein Characterization**

__ Results: __













Don't keep your lane captions in a paragraph format! Should look like this: Lane 1: Lane 2: Lane 3:

__Calculations:__ A-Absorbance E-Molar Absorption (Avg. Absorbance / Concentration x 100) b-Path Length (1 cm) c-Concentration (mol/L)
 * Elution 1 at 280 nm**
 * Beer's Law: A=Ebc**

0.440 = (38850 M-1 cm-1) (1 cm) (c) c = (1.1326 x 10-5 mol/L) (1 L / 1000 mL) (25794.2 g/mol) (1000 mg / 1 g) = **0.292145 mg/mL**

Flow of discussion hard to follow (don't restate what you did in lab) Need to improve your discussion __ Discussion: __ During the starter culture, a single bacteria colony was taken from an agar plate, a medium used to culture bacteria, and put into LB broth, a media used to grow E-coli bacteria with the addition of SOC, super optimal broth, a rich bacterial growth medium. During the transformation process, SOC allowed the bacteria culture to grow in the shaking incubator. During the large culture, more LB broth was added to make the bacteria grow but to prevent uncontrolled growth. The antibiotic ampicillin, was also added into organism BL21(DE3) and plasmid pGEM-gbr22. The cells were ready to harvest when the media turned purple. The media was centrifuged to spin the cells down. Lastly, the PBS stock, a buffer used to keep the pH of the environment stable was used to obtain a homogeneous suspension. At the wavelength of 280 nm, the concentration of elution 1 was calculated to be 0.292145 mg/mL and the yield turned out to be 1.46073 mg. The yield was calculated by multiplying the concentration (0.292145 mg/mL) by the volume (5ml).

Lysozyme was used to break down peptidoglycan (bacterial cell wall) of BL21 (DE3) which made it possible to isolate and extract the pDEM-gbr22 protein. Benzonaze is a nuclease that makes the culture more viscous by digesting the DNA/RNA. The HIS tag system: A DNA sequence specifying a string of six histidine residues is used in the production of recombinant proteins. Expressed His-Tagged Proteins can be detected easily because the string of histidine residues bind to several immobilized metal ions, such as nickel and retains the protein. Sample 1 of contained the original bacterial culture after ~24 hours of centrifugation, sample 2 contained the supernatant of the culture from the lysate, sample 3 contained flow through of resin/buffer after filtration, sample 4 contained loosely bound protein and flow through of Ni-NTA resin with wash buffer, sample 5 contained tightly bound protein with elution buffer 1, and sample 6 contained leftover protein (not removed by elution 1) with elution 2. The wash buffer was a combination of 1xPBS and 20 mM Imidazole while the elution buffers were made with 1xPBS and 250 mM Imidazole. The high difference in concentration of imidazole explains why the wash buffer removes only loosely bound proteins, thus elution 1 contains more proteins than the wash buffer and elution 2. It it difficult to state the size of the protein from the gel because there are 2 bands of equal thickness and darkness present in lane 5, with elution 1. This shows that another protein is present and could've resulted from contamination due to lack of sterile techniques while the wells were being loaded because multiple samples flowed into the other lanes. Because two bands were identified, the purity is only roughly ~50%. There is one band at 25kDa and another at 15kDa but because the protein is expected to weigh around 25kDa in ideal conditions, the protein from the gel is most likely the first dark band.

__ Conclusion: __ The process of overexpressing, purifying, and characterizing pGEM-gbr22 in BL21 (DE3) by filtration, Ni-NTA affinity chromatography, and gel electrophoresis resulted in the calculation of the concentration and yield of the protein, although the exact purity is not known. The gel showed 2 bands of equal intensity which revealed that the protein was contaminated during the run but this error can be reduced in the future via multiple back up trials and more sterile methods. For the future of VDS, these various methods can be implemented on other enzymes in order to target drugs (?) and isolate desired components of a protein. Should have mentioned about enzyme & inhibition essay after further purifying the protein through FPLC

__ References: __ [1] Robichon, C.; Luo, J.; Causey, T. B.; Benner, J. S.; Samuelson, J. C., Engineering Escherichia coli BL21(DE3) Derivative Strains to Minimize E. coli Protein Contamination after Purification by Immobilized Metal Affinity Chromatography. //Appl Environ Microbiol// **2011**, 77(3), 4634-4646.

[2] 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., Protein production and purification. //Nat Methods// **2008**, 5(2), 135-46.