Ar'NiquaR

__Bacterial Protein E.P.C (Expression, Purification and Characterization)__

__Introduction:__ In protein production of E. Coli a good strategy to speed up the process consists of two experiments. First experiment being the expression of a protein in a basic E. coli host strain from a variety vectors with different tags or fusion partners. The second experiment is t he expression of a protein from a standard vector in a number of different //E. coli// host strains. If the protein contains a high number of ** E. coli codons it should be expressed in a strain. ** In protein purification ** gel filtration chromatography ** can be used to increase the purity and homogeneity of the protein sample. The remaining proteins are separated on a porous matrix according to their size and the different molecular weight can be measured using a molecular weight standard ladder. In these 3 labs we were able to express, purify and characterize the bacterial protein and also find the concentration of the purified protein using the Nanodrop spectrophotometer and Beer's Law. Also we were able to find the molecular weight of the characterized protein using the molecular weight standard ladder.

__Materials & Methods:__ For the “Protein Expression” lab materials used were: Ice bucket, 42 C water bath, gas burner, 2x 14 ml clear, sterile round- bottom tubes, 37 C incubator, colirollers, 2 LB Agar Amp plates, 1 spare Agar plate w/o antibiotic, competent cells on ice, plasmid DNA on ice, LB media, SOC media, pipette and pipette tips. Using one spare Agar plate w/out antibiotic this was made the control plate with no DNA, using two LB ampicillin plates one was made the experimental plate with DNA and the other was made the “fun plate” with saliva deposited inside. The next day a single colony was picked and expressed in a large culture in two 125 ml Erlenmeyer flasks after 24 hours in the incubator the culture was centrifuged in the large benchtop centrifuge forming two cell pellets that were resuspended in phosphate buffered saline. For the “Protein Purification” lab materials used were: Ice bucket, beaker of RT (room temperature) water, 1M Imidazole, 10xPBS, 1x PBS, 1.7 ml centrifuge tubes, 2x 10 ml round bottom tubes, 4x 15 ml conical tubes, Bio-rad Econo chromatography column w/ yellow cap, ring stand and clamps, Ni-NTA resin, Benzonase. First step was to lyse the E.coli cells using the Benzonase, then clafify the lysate using the small centrifuge and isolate the soluble fraction by removing the liquid and seringe filtering it to remove extra debris. Then using the Ni-NTA affinity purification the protein was purified and made into elutions 1 and 2. Equipment and Materials from “Protein Characterization” lab were: Mini-PROTEAN electrophoresis tank and lid, power supply and leads, TGS running buffer, Bio-Rad precast polyacrylamide gel, 100 ul 6x gel (or sample) loading buffer, Protein Samples #1-6 from Expression and Purification labs, Molecular Weight standards, plastic container with lid, and Imperial protein stain. First step was to centrifuge all 6 sample tubes and place the samples into the precast gel, then place the gel into the Mini-PROTEAN electrophoresis tank and lid. After 25 minutes removed the gel and stained it using the Imperial protein stain for 1 hour then washed the gel 3 times using nanopure water. The following day using the vacuum dried the gel.

__Results:__ In the “Bacterial Protein E.P.C (Expression, Purification and Characterization)” labs I was able to express bacterial “purple” protein and use that same protein and purify it using the Ni-NTA affinity and then characterize the protein using the mini-PROTEAN electrophoresis tank and Bio-Rad precast gel. Throughout all these different labs I was able to estimate the concentration of the purified protein and also estimate the molecular weight of the charaterized protein.

Figure 1: Control plate from the “Protein Expression” lab that does not contain any DNA to confirm that this was a clean technique and no colonies are growing without the plasmid. Figure 2: “pGEM gbr22” ampicillin plate that contains the bacterial DNA after it has been grown overnight and colonies have formed. Figure 3: “Fun plate” that contains ampicillin and saliva produced by Saurabh Ghosh that has been grown overnight causing bacteria to grow. Figure 4: Two flasks containing the purple culture consisting of LB and ampicillin media, and the pGEM-gbr22 after staying overnight in the shaking incubator to grow. Figure 5: Cell pellet number 1, weighing 0.67 grams, formed after spinning the cells down in the large benchtop centrifuge for 10 minutes.

Figure 6: Cell pellet number 2, weighing 0.87 grams, formed after spinning the cells down in the large benchtop centrifuge for 10 minutes. Figure 7: Elutions 1 and 2 from the “Protein Purification” part of the 3 part lab that consists of the bacterial protein after it had been purified using the combination of batch and column chromatography.

Figure 8: Using the Nanodrop spectrophotometer able to estimate the concentration of the purified protein. Measuring at 280 nm the concentration is 2.13 mg/ml.

Figure 9: Measuring at the maximal wavelength of 574 nm the concentration was estimated to be 2.15 mg/ml using the Nanodrop spectrophotometer. Figure 10: Hand calculations of the concentration using the Beer's Law equation, concentration is 2.19 mg/ml. Figure 11: The Bio-Rad precast polyacrylamide gel after removing it from the Mini-PROTEAN electrophoresis tank, staining it for 1.5 hours on the orbital shaker and destaining it using nanopure water. Figure 12: The Bio-Rad precast polyacrylamide gel after removing it from the vacuum and drying it out. Figure 13: This is the ladder (molecular weight marker used) to measure estimate the MW of the purified protein is about 25 kD.

__Discussion:__ Using the protein expression, purification, and characterization labs I was able to estimate the concentration of the purified protein at two different wavelengths (the maximal wavelength and 280 nm). The estimated concentration of the protein at 280 nm was 2.13 mg/ml and at 574 nm the estimated concentration was estimated to be about 2.15 mg/ml. I was also able to estimate the molecular weight of the charaterized protein to be about 25 kD. The estimated purity would be about 12.5 since the yield is about 50% of the molecular weight. There were a few sources of error in the “Protein Expression” lab I noticed there was a slight difference in the weight of the two cell pellets might have been caused by an uneven distribution of cells in the LB + amp media. And also in the “Protein Characterization” lab I noticed that on the gel the thick purple band that was suppose to appear from sample 1 did not appear but it appeared on samples 2, 3, and 4. We're not quite sure of the reasoning behind this but we can assume that it has something to do the sample 1 and it's transfer from the centrifuge tube into the Bio-Rad precast polyacrylamide gel.

__Conclusions:__ In these 3 labs I was able to express, purify and characterize the bacterial protein and also find the concentration of the purified protein using the Nanodrop spectrophotometer and Beer's Law. I was also able to find the molecular weight of the characterized protein using the molecular weight standard ladder. The concentrations and molecular weight of my protein could be compared with other students from my class that also completed these labs to see if the results were similar. Since I know quite a bit of information about this protein I could search for drugs that could possibly inhibit the purple color.

__References:__ 1.) Hallberg, Martin B. "Protein Purification and Purification." // PubMed.gov // . NCBI, 5 Apr. 2008. Web. 19 Apr. 2011. < [|__http://www.ncbi.nlm.nih.gov/pubmed/18235434__] >.  2.) "Protein Expression and Purification Core Facility - EMBL." //EMBL Heidelberg - The European Molecular Biology Laboratory//. European Molecular Biology Laboratory, 2009-2011. Web. 19 Apr. 2011. . 3.) Spendlove, Ian. "Protocol Online: Protein Purification." //Protocol Online - Your Lab's Reference Book//. Academic Clinical Oncology, 22 Jan. 1996. Web. 19 Apr. 2011. <[]>.