Shashika+R

__**Introduction**__ Purification studies have often been questioned as to the best methods to use. However, purification strategies are now based on past research and evidence. Thus, it is best to go through purification methods used by past researchers and evaluate these based on their relevance and effectiveness. When expressing a protein, it is important to choose either the N or C terminal as a boundary because this affects solubility and expression [1]. Many biomedical labs focus on the folded, globular domains of proteins, which are suitable for expression in E. coli. Many classes of proteins can be integrated into E. coli. The chance of successfully expressing a protein decreases with an increase in molecular weight [2]. Using E. coli as an expression mechanism allows for a fast, cheap test of possible strategies. It is strongly suggested that the protein be produced with a tag, which will help in protein purification. It has been established through research that an N-terminal hexahistidine tag can be removed by a protease that is site-specific [1]. These histidine-tagged proteins can be purified using a simple protocol called immobilized metal affinity chromatography. Also, histidine tags have minimal effect on the characteristics of a protein. The tags also do not have a significant impact on the N-terminal structure of the target. The ultimate objectives of these labs are to over express recombinant pGEM-br22 protein in E. coli, to then purify this protein, and to use gel electrophoresis to analyze the protein samples prepared in the previous two labs. The proteins will successfully be expressed, purified, and analyzed to determine the molecular weight, purity, and final yield of the protein product.
 * Protein Expression, Purification, and Characterization**

__**Materials and Methods**__ Obtain three plates, one experimental plate, one control plate, and one “fun” plate. Add 25 ul of bacteria to each of the two transformation tubes, and use mini-centrifuge to spin. Add 1 ul of plasmid to bacteria in DNA tube. Wait thirty minutes on ice, and place 3 plates in incubator to pre-warm. Heat shock tubes in 42 degree water bath for 45 seconds. Wait two minutes on ice. Add 200 ul of SOC. Shake in incubator for 30 minutes at 37 degrees at 250 rpm. Place 6 colirollers onto each plate. Pipette 50 ul bacteria mixture onto each plate. Roll plate around. Pour off colirollers. Sneeze on “fun plate.” Cover plates with lid, invert, and store in incubator overnight. Grow starter culture in LB supplemented with 100 ug/mL ampicillin. Store plates in 4 degree fridge. Transfer LB to Erlenmeyer flasks, and add ampicillin to makine concentration 100 ug/ml. Add 0.625 ml of starter culture to flasks. Clip into shaking incubator for 16-24 hours. Pour bacteria into conical tube. Run in centrifuge for 10 minutes at 5000 rpm at 4 degrees. Resuspend the cells in a phosphate buffered saline. Syringe filter lysate with 5 ml syringe into 15 ml round bottom transformation tube. Purify protein using batch and column chromatography by adding Ni-NTA resin and bufferto the top of the column and collecting the flow with a round bottom tube labeled “Waste.” Wash Ni-NTA resin with 5 ml of 20 mM imidazole in 1 x PBS, and collect in tube labeled “Wash.”Elute bound protein by adding 5 ml of buffer with 250 mM imidazole. Collect buffer in 15 ml conical tube labeled “Elution 1.” Repeat by adding 5 ml elution buffer and collecting in 15 ml conical tube labeled “Elution 2.” Retain samples. Strip remaining ni-NTA. Use nanodrop spectrometer to record absorbance at 280 nm and maximal wavelength. Add 10 ul of 6 x loading buffer to samples 4-6. Place tubes into heat block at 95 degrees for 5 minutes Remove tubes from heat bloack and centrifuge tubes for 2 minutes at 5,000 rpm. Remoe precast gel from packaging. Place gel cassettes into claping frame, and lock into place. Fill assembly with 1 x TGS buffer. Clear each lane with needle and syringe. Incject buffer into lanes to clear. The first gel is the ladder. Load 20 ul of each protein samples into wells. Run gel for 25 minutes. Remove gel cassette. Add Imperial protein stain to completely cover gel. Place on orbital shaker for one hour. Pour reagent back into container. Add nanopure water and Kimwipe, and leave overnight. Cover gel with saran wrap. Take gel to Biotech lab and place on drying bed. Set temperature to 75 degrees on gradient cycle for 1.5 hours. Turn on vacuum and start cycle.
 * Expression**
 * Purification**
 * Protein Characterization**

__ **Results**: __ Thus, no growth is seen.
 * Figure 1.** Control Plate containing bacteria Bl21 (DE3) and Ampicillin. This plate had bacteria without a vector providing resistance to Ampicillin.


 * Figure 2.** Culture Plate containing bacteria Bl21 (DE3) and Ampicillin, as well as the vector pGEM-gbr22. It is clear that no bacteria grew, though they were supposed to. This may have been caused by non-sterile lab techniques, incorrect addition of DNA to bacteria, or insufficient time for the bacteria to grow. However, a sample was obtained from another group to continue with the experiment.


 * Figure 3.** Plate containing sample swiped from door handle of lab. This plate exhibited no growth after a day, but did eventually exhibit growth.


 * Figure 4**. Bl21(DE3) bacteria in flask. The culture has turned purple because the pGEM-gbr22 protein is being expressed.


 * Figure 5.** The cell pellet remaining after centrifuging bacteria solution. Liquid media was poured out of conical tube, leaving behind the pellet.


 * Figure 6.** Elution 1 and Elution 2, obtained from the last two Buffer purification runs through Econo chromatography column (BioRad). The imidazole allowed the pgbr22 protein to be detached from the Ni-Nta resin. Elution 1 contains a majority of the purified protein.


 * Figure 7.** These two graphs show the absorbance of the purified protein at 280 nm, which were collected by the Nanodrop Spectrophotometer (Thermo Scientific, Wilmington, DE). The first trial yielded an absorbance of 0.261, while the second trial yielded one of 0.693. These average to 0.477. According to Beer's Law, A=Ebc. Thus, with an absorbance of 0.477, an extinction coefficient of 38,850 and a path length of 10 mm, the concentration was determined to be 1.2278 E-5M. The concentration was then multiplied by the molecular weight of the protein (found online). The concentration was found to be 0.3167 mg/ml.


 * Figure 8.**These two graphs show the absorbance at the maximal wavelegnth of 574 nm. The absorbance readings were 0.036 and 0.055, with an average of 0.0455. The relative max at 574 nm illustrates that the maximal wavelegnth occurs at that point. The path length for this sample was 1 millimeter. According to Beer's Law, A=Ebc. Withan absorbance of 0.0455, an extinction coefficient of 118,300 and a path length of 1 mm, the concentration was determined to be 3.846E-6M. The concentration was then multiplied by the molecular weight of the protein (found online). The concentration was found to be 0.0992 mg/ml.


 * Figure 9.** W et gel obtained from gel electrophoresis of Samples 1-6, which contained the purple protein of interest, gbr22. Lane 1 contains the MW standard (Fermentes, SM0671), lanes 2-7 contain Samples 1-6 (lab partner's), and lanes 8-10 contain a second set of Samples 4-6 (mine). Because of the 3 lines in Sample 5, it appears as though the solution was not very pure.


 * Figure 10.** Fermentas PageRuler Molecular Weight Standard, SMO671. The ladder was used as a reference to compare wet gel to. Through comparison it is evident that molecular weight of protein was about 25 kDa.

__ Discussion: __ Firstly, the recombinant protein, pGEM-gbr22, was over-exposure in E. coli by inserting the plasmid into the bacteria. The bacteria were then placed on ice, then heatshocked in a 42 degree water bath, then incubated at 37 degrees Celsius. The starter culture was then grown in LB broth with 100 ug/ml ampicillin, where it was transferred. The tube containing the culture was then incubated for eight hours. This starter culture was then transferred to another Erlenmeer flask, containing LB broth and ampicillin. This culture was placed in the shaking incubator overnight. After the culture was purple, the centifige (Allegra X-15) was used to harvest cells. Sample 1 was taken from this culture. The bacteria was poured into a conical tube with the media, and centifuged. The purple pellet was saved, while the liquid was decanted. 1 x PBS was added to the conical tube containing cell pellet. The tube was vortexed to obtain an even suspension. Lysozyme was then added and vortexed again. The Lysozyme was added to damage the peptidoglycans in the bacterial cell wall of // E. Coli, //essentially killing the bacteria.

The next stage of the lab was to purify the protein expressed in bacteria earlier. The conical tube was incubated for 20 minutes to ensure lysis of the cells. Benzonase was added to conical, with purpose of reducing viscosity by digesting DNA in mixture. Lysate was distributed into microcentrifuge tubes and centrifuged for 20 minutes. Sample 2 was extracted from this upernatant. A micropipettor was used to transfer liquid supernatant, leaving cell debris behind. The lysate was then syringe filtered. The Bio-Rad chromatography Econo column was set up.Because the protein was modified to have histidine residues added to the C-terminus, it could be separated from other cellular proteins. The histidine residues bind to cations like nickle, and thus be released from the Ni-NTA agarose through imidazole. This is the basis of purification. The Ni-NTA resin and buffer was transferred to the top of the column. The flow was collected in a 10 mL tube labeled "Waste." Sample 3 was collected from this. The wash step removed proteins loosely bound to the resin. The Ni-NTA resin was washed with imidazole in PBS, and allowing the flow to be collected in round bottom tube labeled "Wash" Sample 4 was collected from this. The gbr22 protein was released from the resin by using imidazole. The buffer was added and then collected in a conical tube labeled Elution 1, from which Sample 5 was taken. The buffer was added again and taken up in a conical tube labeled "Elution 2." The wash buffer contained considerably less imidazole than Elution 1, so it removed proteins less tightly bound to the nickle resin. The Nanodrop spectrophotometer was then used to estimate the concentration of the final purified protein. At 280 nm, the absorbance averaged out to 0.477. The concentration of the purified protein at 280 nm was found to be 0.3167 mg/ml. The wavelegnth in the visible region where the protein absorbs maximally was determined to be 574 nm, taken from online. The concentration of the purified protein was found to be 0.0992 mg/m at this maximal wavelegnth.

Lastly, gel electrophoresis was used to analyze protein samples. The gel samples were prepared by adding 6 x loading buffer to samples collected in previous experiments. Tubes were then placed on heating block. After electrophoresis module was assembled, samples were loaded and gels were run for 25 minutes. The gels ultimately were compared to standard, and it was determined that the elution (run 5) had MW of 25 kDa, and minimal purity due to the multiple bands.

Source of error: The spectrophotomer may have been incorrectly calibrated, altering absorbance reading. When distibuting samples in wells, the gel may have been broken by the syringe, altering the run. The bacteria may have been contaminated, causing foreign growths in the samples. Human errors in accuracy may have greatly altered the data.

__ Conclusions: __ In conclusion, this protein lab contained three main parts, expression, purification, and characterization. In the expression phase, a recombinant protein was overexpressed in bacteria using transformation. In the purification phase, the Histidine tags were utilized to attach to the nickle resin, and then detach the protein using imidazole. The protein characterization phase utilized gel electophoresis to further analyze the samples yielded in the earlier phases of the lab. Though the protein was purified, the electophoresis revealed that the protein still wasn't completely pure. In the future, proteins can be purified and characterized from other sources, such as viruses and bacteria, and analyzed to find drugs which will target their production and overall function.

__ References: __ 1. Nat Methods. **2008**Feb;5(2):135-146. Protein Production and Purification. (accessed April 16, 2012) 2. Amersham Biosciences. //Protein Purification Handbook//. []. (accessed April 16, 2012)