NICOLET+F


 *  __The__ **__** purification and characterization of pGEM-gbr22 expressed by E. Coli. **__

  Intro should have more background info on theory or on the technique than restating methods __Introduction: __  The use of recombinant protein is commonly used in the study of biological sciences, producing a pure targeted protein [1]. Protein production and purification are important for the characterization of proteins in that it allows for the amplification and isolation of the single target protein. Lysate is used to harvest the protein from the cell. The overexpressed protein is then bound to nickel ions on the Ni-NTA resin. The histidine residues are also attached to the Ni-NTA resin. This allows for an efficient purification process since the matrix components are unable to flow through the chromatography column. A gel electrophoresis can then be run to determine the size and purity of the protein sample [2]. Three labs consisted of expressing, purifying and characterizing the recombinant protein, pGEM-gbr22.

__Materials & Methods: __ Separate M&M into sections (protein expression, protein expression, and protein characterization) In preparation, 3 plates were made and labled (1 experimental with DNA, 1 control without DNA and 1 "fun plate," which was coughed on). Transformation tubes were prepared for each plate, each containing 25 ul of bacteria (need to be specific here (ex. BL21(DE3)/ also need to include company & state)) , by heat shocking at 42°C for 45 seconds and returning to the ice bucket for 2 minutes. 200 ul of SOC media was added to each tube and placed in the shaking incubator at 37°C for 30 minutes at 250 rpm. The mixtures from each tube were transferred to their appropriate plates and placed in the 37° incubator overnight. A colony of bacteria from the DNA plate was transferred to LB/ampicillin media to grow in the shaking incubator for 8 hours at 37°C and 250 rpm. 0.625 mL of the culture was transferred to 25 mL of 100 ug/mL ampicillin in LB solution and stored in the shaking incubator for 24 hours at 37°C and 250 rpm. This bacteria was centrifuged to obtain a 0.40 g pellet of the protein and 2.5 mL of 1xPBS was added and vortexed. 51 ug of lysozyme was added. 2 ul of cyanase (used benzonase) was added and incubated for 15 minutes and then centrifuged for 20 minutes. The soluble fraction was filtered through a 0.22 syringe filter PES with a 5 mL syringe and purified using a combination of batch and column chromatography. NiNTA resin buffer was used as the 1st rinse through the bio-rad and collected as “Waste”. A wash buffer containing 0.2 mL imidazole was used for the 2nd rinse and collected as “Wash.” An elution buffer containing 2.5 mL imidazole was used for the 3rd and 4th rinse and collected as “Elution 1” and “Elution 2.” The Nanodrop spectrophotometer was used at 280 nm and 574 nm (the maximum absorbance wavelength), along with the extinction coefficient and Beer’s law, to find the concentration and yield of the protein in “Elution 1”. Samples for each step in the process of purifying were collected and labeled for a total of 6 samples. Sample 1 was centrifuged for 5 minutes at 5,000 rpm to retain the pellet, which was suspended in 200 ul of water and added to 40 ul of loading buffer. 10 ul of loading buffer was added to Samples 2 to 6. All samples were placed into a heat block at 95°C for 5 minutes and centrifuged for 2 minutes at 5,000 rpm. The electrophoresis module was assembled and filled with 1xTGS buffer. 20 ul of each sample was loaded into the 10 lanes of the gel in the following order: MW, Sample 1, Sample 2, Sample 3, Sample 4, Sample 5, Sample 6, Sample 4 from 2nd student, Sample 5 from 2nd student, Sample 6 from 2nd student. The gel electrophoresis was run for 20 minutes at 200 V. The gel was washed 3 times for 5 minutes each with nanopure water in the orbital shaker, stained with the imperial protein stain for 1.5 hours in the orbital shaker, and then rinsed 2 more times with nanopure in the shaker. The gel was then stored in new nanopure water with a kimwipe in the orbital shaker overnight. The gel was dried on Whatman filter paper, covered with cellophane, in the drying bed at 75°C on Gradient cycle for 1.5 hours using the vacuum. The gel was now able to be analyzed.

Need to improve on captions (what kind of plate is it?)



 Beer's Law is A=Ebc. Inputting the absorbance value, the extinction coefficient and the cuvette path length, the concentration can be found. This equation looks like 0.332 = (38850)(1)(c). (need to include units) The concentration is found to be 8.546e-6 Mol/L. Multiplying this concentration by the molar weight of the protein (25794.2 g/Mol), the yield can be found as 0.2204 mg/mL. Since there is 5 mL, the final yield is 1.102 mg.





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

Weak analysis (Ex. need to talk about the importance of using LB/Amp plates) __Discussion: __ During the protein expression portion of this lab, a cell pellet of 0.40 g was collected after a harvested culture was centrifuged. In order to purify the protein, lysozyme was used to cause the cells in which the protein was contained to burst. Cyanase (used benzonase)  was then used to extract the DNA from the cell. The Bio-rad chromatography column allowed for purification of the protein, as it was too large to pass through being bound to the nickel which was bound to a complex during the wash. Once the wash was complete, the elution buffer was run through, breaking the HIS tag bond (where does the 6x His tag reside on the protein sequence)  between the protein and the Ni complex. This allowed to protein to fall through into “Elution 1” and “Elution 2,” keeping still too large Ni and it’s complex in the column. The difference between the wash buffer and the elution buffer is that the concentration of imidazole, a competitive inhibitor for Ni with the protein, is higher in the buffer. After purifying the protein and using the Nanodrop spectrophotometer, it was found that the maximum absorption wavelength is 574 nm. Using Beer’s law, the concentration of the protein was discovered to be 8.546e-6 Mol/L and the yield was found to be 0.2204 mg/mL. The protein was characterized using the 6 samples taken from various parts of the lab. Sample 1 contained the protein culture was was harvested. Sample 2 contained the lysate from the Cyanase enzyme. Sample 3 contained the flow of waste solution collected from the Ni-NTA purification. Sample 4 contained a flow of wash solution collected from the Ni-NTA purification. Sample 5 contained the the first elution buffer flow. Sample 6 contained the second elution buffer flow. Once characterized, the molecular weight of the protein was estimated to be 25 kDa. The molecular weight determined in the protein purification lab was 25794.2 D, which is very close to the weight estimated. In the “Elution 1” lane, there were 3 bands of protein present. If the protein was 100% pure, the expressed protein would be the only band shown in this lane. Because of this, it is known that there is a small amount of other types of protein in this sample. Sources of error include running the wash and elution rinses wrong. My partner and I ended up joining another group after realizing we had combined the wash and elution flows. (use 3rd person)

__<span style="background-color: white; font-family: Cambria,serif;">Conclusions: __ <span style="background-color: white; font-family: Cambria,serif;"> This lab was a multi-step process which was split into 3 labs consisting of expressing a bacterial protein so that it was able to be purified and characterized through gel electrophoresis. Throughout the course of the labs, samples were collected and compared in the gel electrophoresis characterization. This process of expressing, purifying and characterizing a protein is commonly used in the field of biological sciences. These techniques can further be used in future virtual drug screening wet labs to ultimately discover new compounds to act on these proteins and work as drugs. Need to talk about enzyme & inhibition assay

__<span style="background-color: white; font-family: Cambria,serif; font-size: 13px; line-height: 1.5;">References: __ <span style="background-color: white; font-family: Cambria,serif; font-size: 13px; line-height: 1.5;">[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., Protein production and purification. //<span style="font-family: Cambria,serif; font-size: 13px; line-height: 1.5;">Nature Methods //**<span style="font-family: Cambria,serif; font-size: 13px; line-height: 1.5;">2008, **//<span style="font-family: Cambria,serif; font-size: 13px; line-height: 1.5;">5 //, 135-46.

<span style="font-family: Cambria,serif;">[2] European Molecular Biology Laboratory. Protein Expression and Purification Core Facility Protein Purification. Need to improve how your reference your sources