Landon+K

__ Expression, Purification, and Characterization of pGEM-gbr22 __ = =

Many scientific research facilities have come to use recombinant proteins in their lab daily. With each individual protein, proper procedures to express, purify and characterize the given protein must be decided among the ample combinations of methods. The use of E. coli BL21(DE3) as an expression host for recombinant proteins is a great choice due to its ability to express a wide range of proteins. It is also very convenient to obtain and has a relatively low cost [1]. Using a marker for ampicilin resistane is often used to recognize the success of transformed colonies that will be used to grow a starter culture. Then the initiation of protein expression and harvesting of the cell pellet can occur [2]. This experiment will entail the transformation of the E. coli BL21(DE3) with a plasmid vector encoding gbr22 (purple protein), expressing the protein in a large culture, and successive purification and characterization of the gbr22 protein. Concluding this experiment should result in high concentration and purity of the protein.
 * __Introduction__ **

A cylindrical tube containing 25 µl of competent //E. coli//BL21(DE3) cells(New England BioLabs) and 2 µl of pGEM-gbr22 was left on ice for 30 minutes, heat shocked in a 42°C water bath for 45 seconds, put on ice for 2 minutes, had 200 µl of SOC media added, and was put in a 37°C shaking water bath for 30 minutes at 250rpm. The bacteria was plated on dishes of agar and stored in a 37°C incubator overnight. A culture tube containing a transformed bacterial colony, 10 µl of ampicillin and 5 mL of LB was prepared and placed in a shaking incubator at 37°C at 300rpm for 8 hours. A flask containing 25mL LB, 50µL of ampicillin, and 625µL of starter culture was placed ina shaking incubator at 37°C at 300rpm for 20 hours. 500 µL of the culture was saved as Sample 1 and the rest was centrifuged for 10 minutes at 5000rpm at 4°C. The pellet was resuspended in 2.5 mL of 1x PBS and 50 µL of lysozyme, and was stored in a -20°C freezer.
 * __Materials & Methods:__ **

The cell suspension was thawed, had 2µL of Benzonase added, and was microcentrifuged for 20 minutes at 14000 rpm at 4°C. 50 µL of the supernatant was saved as Sample 2 and the rest was disposed. The lysate was filtered through a syringe filter and had .5 mL ni-NTA added. The mixture was added to a chromatography column (Bio-Rad), then Wash Buffer, then Elution Buffer twice. 50 µL of flow through after each step was collected as Samples 3-6. The Nanodrop spectrophotometer was used to measure absorbance of the purified protein at 280 nm and 574 nm. Sample 1 was microcentrifuged for 5 minutes at 5000rpm and resuspended in 200 µL of water and 40 µL of loading buffer. Samples 2-6 had 10 µL of 6x loading buffer added. All samples were put on heat block at 95°C for 5 minutes and centrifuged for 2 minutes at 5000rpm. MW standards(Fermentes, SM0671) and Samples were loaded into wells of gel(Bio-Rad) and gel was run for 25 minutes at 200V. The gel was removed, stained with Imperial protein stain, washed for 12 hours, and dried on Whatman filter paper on a drying bed for 75°C for 1.5 hrs.

  Figure1. Starter culture containing BL21 (DE3) and Amp (plate containing pGEM-gbr22 shown on the right & Control plate shown on right)
 * __Results:__ **

Figur 2. Large culture of BL31(DE3) & pGEM-gbr22 grown in LB and Ampicilin in a shaking incubator at 37 degrees Celsius and 200-350 rpm

Figure 3. Cell pellet from the large culture after being centrifuged for 10 minutes at 5,000 rpm and 4 degrees Celsius

Figure 4. Elution 1 (left) and Elution 2 (right) containing purified pGEM-gbr22 protein washed by Elution buffer solution from a Ni-NTA column using 250 mM imidazole and 1 M PBS solution.

Figure 5. Nanodrop spectrophotometer results of Protein A280 setting with Elution #1 containing purified gbr22 protein Trial #1

Figure 6. Nanodrop spectrophotometer results of Protein A280 setting with Elution #1 containing purified gbr22 protein Trial 2

Figure 7. Nanodrop spectrophotometer results on UV-Vis Mode with Elution #1 containing purified gbr22 protein Trial #1

Figure 8. Nanodrop spectrophotometer results on UV-Vis Mode with Elution #1 containing purified gbr22 protein Trial #2

** Beer's Law Calculation ** Determining Concentration of Protein using 280nm Molecular weight: 25794.2 g/mol Extinction Coefficient: 38850 M^-1*CM^-1 A=Ebc A=(.898+.868)/2 =.883 .883= c (1) (38850 M^-1*CM^-1) c=.00002273 mol/L Determining Concentration of Protein using Maximal Wavelength A= (.124+.127)/2 = .1255 .1255 = c (1) (118,300 M^-1*CM^-1) c= .00000106 mol/L ** Nano **** drop Spectrophotometry ** 280nm wavelength: .00002775 mg  (.0002273mol/L)(25794.2g/mol)= __ .00000555 g/L __ .00000555 mg/ml (5ml)= .00002775 mg  Maximal wavelength: __. ____ 13682046 __ mg ____ (.00000106mol/L)(25794.2g/mol)= .02736409 g/L (.02736409mg/mL) (5mL) = .13682046 mg

Figure 9. Result of Gel Electrophoresis using mini-PROTEAN of Samples taken during Purification Step for gbr22 (right to left: MW-1-2-3-4-5-6-[4-5-6 sample from partner])

Figure 10. Ladder for Fermentas of MW's __** Discussion **__ The MW of the purified gbr22 protein was estimated to aproximaitly be 28kDa, using the MW standards (Fermentes, SM067) as a reference point. Due to the presence of three other protein bands in the Sample 5 lane of the gel electrophoresis, it was concluded that gbr22 was not completely pure. It was estimated to be at a 25% purity. In order to release the desired soluble protein, Lysozyme was added to digest the cell walls and benzonase was added to digest the DNA/RNA. Sample 1 contained bacterial starter culture (in log phase). Sample 2 contained soluble fraction of the lysate. Sample 3 contained flow through waste. Sample 4 contained washed protein solution. Sample 5 had eluted protein solution. Sample 6 had protein solution eluted twice. The elution buffer was a higher concentration of imidazole to allow for more protein to be released. This system involves fusing a histidine affinity tag, or number of histidine residues, to a protein. This allows for the purification of the protein with an immobilized metal affinity chromatography techniqe [1]. Any source of error includes contamination, miscalculations, and/or imperfections of readings, solutions, and/or measurements.

During this experiment, overexpression of recombinant gbr22 protein in E. coli occurred. The protein was later purified through a series of filtrations, and analyzed through gel electrophoresis and a Nanodrop spectrophotometer. The gbr22 protein was carefully purified with little intrusion from extra materials left from the cell. This was verified in the outcome of the gel electrophoresis. Techniques gained in this lab will be used when a specific protein is needing to be expressed or isolated for any reason.
 * __Conclusion __**

**__References__** [1] Nat Methods. Protein production and purification. **2008**, //5// ( //2// ):135-46.

[2] European Molecule Biology Laboratory. Protein Expression and Purification Core Facility. [] (accessed April 14, 2012)