ANITA+V

__ Title: __ ** Expression and Purification of pGEM-gbr22 Fluorescent Purple Protein ** Make intro more relevant to this lab Intro should be more background info on theory or on the technique than restating methods Some assertions in discussion are incorrect

__ Introduction: __ Protein expression and purification has been widely utilized in the biological sciences for the study of proteins [1]. Because proteins are known to play vital functions in cell processes, protein characterization is especially important in the biopharmaceutical industry [2].

In the past, only highly skilled experts in the field possessed the ability to carry out these techniques; however, because of increasing technology, these methods are now much more common. Due to the unique nature of each protein, it is difficult to create a systematic method for protein purification [1]. Some factors that can affect the result of protein purification include lysis method, buffer composition, temperature, and protein solubility [3]. Therefore, different steps will likely be taken for different proteins [1].

The purpose of this lab is to express protein in //E. coli// BL21(DE3) (Cite company & state) cells that is coded for by the pGEM-gbr22 plasmid, to purify the gbr22 protein, and to analyze the purity of the final protein sample. We hypothesize that BL21(DE3) will be transformed with the pGEM-gbr22 plasmid and express the fluorescent protein this plasmid codes for; moreover, following purification, gbr22 will be the only protein in the sample.

__ Materials & Methods: __ An experimental and control Agar plate contained LB media and Ampicillin, whereas the ‘fun’ plate contained media but no antibiotic. Two transformation tubes were each filled with 25 ul of //E. coli,// BL21(DE3). 300 ng of plasmid DNA (pGEM-gbr22) was added to the experimental transformation tube. Following icing and a 45 second heat shock, 200 ul SOC media was added before incubation. 50 ul of each bacteria/SOC mixture was transferred to each plate. The ‘fun’ plate was coughed on.

Following incubation, starter cultures were prepared by adding 0.01 mL of 50 mg/mL ampicillin to sterile culture tubes with 5 mL LB. Bacteria was transferred from experimental Agar plate colonies into the tubes and stored in a 4°C fridge. Once the bacteria were in log phase growth, 0.625 mL of the starter culture was transferred to a 125 mL flask with media and incubated.

Once turbid and purple, the cultures were centrifuged (Which centrifuge did you use) and only the cell pellet was retained. The cells were resuspended in 1X PBS, 51.02 ul of 50 ug/ul lysozyme was added, and the tube was stored at -20°C.

To purify the protein, 2 ul of cyanase was added. After incubation, the cells were centrifuged and the soluble supernatant was isolated into a clean tube. The lysate was filtered through a 0.45 um Millex syringe filter. Column chromatography was then utilized with Ni-NTA beads. The protein was washed with 20 mM imidazole and eluted with 250 mM imidazole. A Nanodrop spectrophotometer (Cite company & state) was used to measure protein concentration at 280 nm and 574 nm.

Six samples collected during the purification process were run through gel electrophoresis with PageRuler Prestained Protein Ladder (ThermoScientific, #26616) as the molecular weight standard. Purity of gbr22 was determined.

__ Results: __ Figure 1a: BL21(DE3) bacteria with LB+Amp media grown at 37 degrees Celsius. Approximately 100 colonies visible. (Specify no DNA control plate) Figure 1b: BL21(DE3) bacteria transformed with pGEM-gbr22 plasmid with LB+Amp media grown at 37 degrees Celsius. 10-15 colonies visible. Figure 1c: Plate that has been coughed on with LB media grown at 37 degrees Celsius. No colonies visible. (Specify no Amp)

Figure 2: BL21(DE3) bacteria transformed with pGEM-gbr22 plasmid after incubation in log phase growth period. Purple color signifies expression of protein coded by pGEM-gbr22.

Figure 3: Cell pellet of BL21(DE3) bacteria transformed with pGEM-gbr22 following centrifuging to isolate cells from media. Purple color signifies expression of protein coded for by plasmid. Mass: 0.37 g.

Figure 4: Elution 1 buffer with protein gbr22. 5 mL obtained.

Figure 5: Elution 2 buffer with protein gbr22. 5 mL obtained.

Figure 6: Absorbance vs. wavelength Nanodrop spectrophotometry reading for Elution 1 at wavelength=280 nm. Trial 1. Protein is gbr22.

Figure 7: Absorbance vs. wavelength Nanodrop spectrophotometry reading for Elution 1 at wavelength=280 nm. Trial 2. Protein is gbr22.

Concentration at 280 nm: Average A: (0.239+0.194)/2= 0.2165 c= 0.2165/[(38850 M-1 cm-1)(1 cm)]= 5.57*10^-6 mol/L (25,794 g/mol) = 0.144 mg/mL

(Yield calculation?)

Concentration at 574 nm: Average A: (0.190+0.230)/2= 0.21 c= (0.21)/[(118300 M-1 cm-1)(1 cm)]= 1.775*10^-7 mol/L (25,794 g/mol)= 0.0458 mg/mL

Figure 8: Electrophoresis gel with Samples 1-6 and samples 3-6 in water. Molecular weight standard: PageRuler Prestained Protein Ladder.

Figure 9: Electrophoresis gel after drying. Samples in each lane are labeled. Molecular weight standard: PageRuler Prestained Protein Ladder.

Figure 10: Molecular weight standard used for gel electrophoresis: PageRuler Prestained Protein Ladder (Product #26616)

__ Discussion: __ The pGEM-gbr22 plasmid contains an ampicillin-resistant gene, so colonies of BL21(DE3) that were successfully transformed with the plasmid were selected for in the LB+Amp plate (Fig 1b). Once the cells were harvested, lysozyme was added in order to digest the cell walls. Cyanase was added in order to digest the DNA/RNA of the //E. coli// cells. The 6-histidine tag assisted in the protein purification process. This tag is much smaller than other tags, so it could be utilized to label the protein without interference to the gbr22 structure or function. The histidine tag was also not very immunogenic and was conformation independent. When gbr22 was added to the Ni-NTA beads in the column, the 6xHIS tag bound tightly to the Ni2+ ions and formed a resin with the beads (doesn't form resin, just binds to the beads. resin is just another term for the beads), allowing other cell waste materials to flow through the column. During the Wash buffer step, a 20 mM imidazole solution was used to compete with and release proteins that were bound less tightly to Ni2+. These loosely bound proteins were likely proteins that contained histidines, but not a 6-histidine structure like gbr22. The elution buffer was a strong 250 mM imidazole solution that competed with the gbr22 protein to bind with Ni2+ and in effect, release gbr22.

According to Nanodrop spectrophotometry at 280 nm (a wavelength at which most proteins absorb), there was 0.144 mg/mL of protein. According to the reading at 574 nm (at which only gbr22 absorbs), there was 0.0458 mg/mL protein. This indicated that there may be other proteins in the sample. The results from electrophoresis showed 1 extra thick band, and 1 thin band, for approximately 40% purity. The size of the protein was 25,794 g/mol and during electrophoresis, the protein was determined to be ~25 kDa, so the results for size were relatively consistent.

Six samples were run through the gel. Sample 1 was taken after adding lysozyme to the cells. Sample 2 was taken from the soluble fraction following the addition of cyanase and centrifuging of the cells. Sample 3 was the taken from the waste material after initially allowing the lysate to flow through the column. Sample 4 was taken from the waste material following the Wash buffer step. Sample 5 and 6 were the purified protein samples following the Elution 1 and 2 steps.

A source of error may have arisen in contamination when initially plating BL21(DE3). The control plate shows several colonies, which indicates that aseptic technique may not have been optimal in the experimental plates. Because there are very few colonies on the experimental plate and abundance on the control plate, the ampicillin on the control plate may not have been distributed properly, causing the error. The scarce colonies on the experimental plate suggest error during transformation with gbr22 plasmid. A source of error during spectrophotometry may be the uneven distribution of protein in solution, accounting for differences in absorbance values during different trials. Lastly, there was error in observing an extra protein in the gel. Several other experimenters encountered this same extra protein, indicating contamination of the pGEM-gbr22 plasmid sample itself. (Not contamination of the plasmid, contamination of the purified protein/Elution 1)

__ Conclusions: __ The protein gbr22 was expressed in BL21(DE3) and isolated using purification techniques. Following purification, spectrophotometry was utilized to determine the concentration and purity of the sample, and these results were compared with results from gel electrophoresis. In drug research, these methods can be utilized to produce and purify target proteins for further experimentation, while verifying that these proteins are truly pure samples.

__ References: __ [1] Acton, T.B.; Albeck, S.; Almo, S. C.; Anderson, S.; Arrowsmith, C.; Atwell, S., Protein production and purification. //Nat Methods// **2008**, 5, (2), 135. [2] European Molecular Biology Laboratory. Protein Expression and Purification Core Facility. [] (accessed April 17, 2013). [3] Arbab, A.S.; Varma, N.R.S., Utilizing protein purification techniques to characterize protein structure and function. //The Internet Journal of Microbiology// **2010**, 8, (2).