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= Lab Protein Expression =

The Characterization of Protein through Bacterial Transformation and Protein Purification

Introduction: Recombinant proteins are used throughout biology and the biomedical sciences. Their ability to be widely and technologically produced allows for greater knowledge and understanding in these areas. These proteins are able to be expressed in bacteria, yeast, and even insect or human cells. As far as purification, researchers have the ability to control selectivity by including low concentrations of imidazole in chromatography buffers, and even through the utilization of common resins with slightly different binding capacities and binding strengths. Also, controlling the ratio of recombinant protein to the column size can optimize the final purity of the protein. It is beneficial to determine the amount of the soluble target protein to be loaded on the column and this can be estimated from small-scale expression trials. Protocols have been put in place for protein purification, which have proven reliable and effective in extractive purified proteins. (Nat Methods, 2008) In the following labs, protein gbr22 was expressed through bacteria. The protein was then extracted and purified and later characterized on a gel and compared to a ladder protein.

Materials and Methods: The materials required for the three labs include an ice bucket, a 42-degree water bath, a gas burner, two 14 mL clear, sterile round-bottom tubes, a 37-degree incubator, colirollers, 2 LB Agar AMP plates, 1 spare Agar plate without antibiotic, competent cells on ice, LB media, SOC media, pipette, and pipette tips. In addition, it was necessary to obtain a beaker of RT water, 1M Imidazole, 10X PBS, a 1.7mL centrifuge tube, two 10 mL round-bottom tubes, four 15 mL conical tubes, a Bio-rad Econo chromatography column, ring stand, clamps, Ni-NTA resin, and Benzonase. Also, the labs require a Mini-PROTEAN electrophoresis tank and lid, a power supply and leads, TGS running buffer, Bio-Rad precast polyacrylamide gel, 100 uL 6X gel loading buffer, protein samples #1-6 from Expression and Purification labs, plastic container with lid, and Imperial protein stain. In order to start the lab process, 25 uL of the competent bacterial cells were added to the tubes. Plasmid was added to the DNA tube only. This was followed by a wait time of 30 minutes on ice. The tubes were then heat-shocked in 42-degree water for 45 seconds and then put back on ice for two minutes. Following, 200 mL of SOC media was added and then the tubes were shaken in the incubator for 30 minutes at 37 degrees and 250 RPM. Next, 50 mL of the bacterial SOC were transferred from the tubes onto each plate and rolled around using colirollers, which were removed afterwards. The plates were covered and inverted for storage in the 37-degree incubator overnight. The following day Ampicillin was added to the two tubes along with 5 mL of LB. The pipette tip was used to pick a strand of bacteria to swirl into the tube. The tubes were then placed into the shaking incubator. Plates were wrapped in parafilm and stored in the 4-degree fridge. Later, 25 mL of LB and Ampicillin were added to flasks along with 0.625 mL of the starter culture. These were then secured in the shaking incubator. The next day, 500 uL of the sample was placed in an Eppendorf and stored in the 4-degree fridge. Bacteria was poured into a conical tube and centrifuged for 10 minutes at 5,000 RPM. The liquid was decanted and 2.5 mL PBS was added to the tube and vortexed. Then, lysosome was added and vortexed followed by storage in the -20-degree fridge. For purification, a 2.5 mL suspension was thawed and incubated. Two uL of Benzonase were added and then the lysate was put into several microcentrifuge tubes and centrifuged for 20 minutes. Afterward, a 50 uL sample was taken and labeled as Sample 2 and stored. Using a P1000 pipette, the supernatant was transferred to a clean conical tube, leaving the cell pellets behind. The lysate was syringe filtered into a round-bottom tube. 10X PBS and 1M Imidazole were added as well as nanopure water and the mixture was vortexed. 0.5 mL of Ni-NTA resin was added and then incubated. A Bio-Rad colum was set up and then the resin and buffer were transferred to the column. Clear flow was collected and labeled as waster. Then a 50 uL sample was taken and labeled as Sample 3. The Ni-NTA resin was washed with Imidazole and PBS by adding the buffer to the top of the column. A 50 uL sample was taken and labeled as Sample 4. 5 mL of the buffer was added, followed by two collections which were labeled Elution 1 and Elution 2. A 50 uL sample of each elution was taken and labeled as Sample 5 and Sample 6. The Ni-NTA was then cleaned appropriately. Protein characterization required preparing Samples #1-6 for gel loading. The electrophoresis module was assembled and then a precast gel was made using the Samples from purification. The gel was then removed afterward and cleaned with distilled water and an orbital shaker. The gel was stained with Imperial protein stain and left overnight. Once the protein stain had been washed off, the gel was dried after being placed on Whatman paper and covered with Saran wrap. The process took one and a half hours.

Results:  Figure 1. Digital representation of the E. coli BL21 (DE3) with the plasmid DNA. It is shown here as grown on an LB agar plate with antibiotic to select for colonies that have the gene for antibiotic resistance. The purple tint of the bacterial colonies signal that the bacteria has "picked up" the plasmid DNA.   Figure 2. Digital representation of the E. coli BL21 (DE3) without the plasmid DNA. It is shown here as grown on an LB agar plate with antibiotic; however, without the plasmid DNA, the bacteria lacks the gene for antibiotic resistance, and thus there is no visual indication that the bacteria is growing and thriving.   Figure 3. Digital representation of the large culture made for overnight expression. The bacteria has been incubated in an LB and Ampicillin media overnight. Since DNA uptake in bacteria is not 100% efficient, it is important to get rid of the bacteria that did not obtain the Ampicillin resistance from the plasmid DNA; thus, the mixture with the Ampicillin/LB media left only the bacteria colonies that were transformed by the plasmid DNA and obtained that antibiotic resistance.  Figure 4. Digital representation of the wet pellet made by centrifuging the bacterial solution in conical tubes. The LB/Amp media was decanted after the centrifugation so that all that remained was the wet pellet of bacteria cells. The weight of the first pellet was 0.56 g and that of the second pellet was 0.57 g.  Figure 5. Digital image of elution one made in the protein purification lab. The purple color shown represents the lesser purified protein of the two elutions. Six milliliters of solution were gathered in this elution.   Figure 6. Digital image of elution 2 made in the protein purification lab. The clear color shown represents the most purified protein solution of the two elutions. Five milliliters of solution were gathered in this elution.  Figure 7. Digital representation of the Absorbance graph of the first elution made in the protein purification lab. The measurement was made at the wavelength of 280 nm. Figure 8. Digital representation of the Absorbance graph of the first elution made in the protein purification lab. The measurement was made at the wavelength of 574 nm. This wavelength was found to be the maximum wavelength for the protein, GBR22, used in this lab. Figure 9. Digital representation of the gel made in protein characterization. The bottom column represents the ladder, or molecular weight marker, and the following columns represent the protein, by bands, and other cellular components by the rest of the purple striations. Figure 10. Digital representation of the protein ladder used as a molecular weight marker in the protein gel. The scale gives readings from about 10 kDa to about 170 kDa. Beer’s Law Calculations from Protein Purification: Concentration 1 4.84 = (38,850) (.1) C   C = .0012458 M    Concentration 2 8.15 = (118,300) (.1) C   C = .00068893 Discussion: From the initial observation of the Agar plates, it was deemed that DNA was necessary for the bacteria transformation, followed by the uptake of the protein trait and replication. Figure 1 and the presence of purple bacterial colonies indicate this. Without DNA, the bacteria have no method or pathway of replication for the protein production, as shown by the empty Agar plate in Figure 2. The purple colors in the flask mixture and in the pellets in Figures 3 and 4, illustrate that the protein is in fact present, since gbr22 is associated with purple luminescence. The two different elutions in Figures 5 and 6 demonstrate that the protein purification was successful. This is illustrated by the change in color of the mixture, from purple to clear. Since the mixture was lysated, the cells of the bacteria were destroyed and the protein was released. The purple in the first elution represents the excess cell parts and organelles no longer associated with the protein. The lack of purple in the second elution demonstrates that there are no more waste products in the mixture and that protein is all that remains. Nonetheless, when the gel is analyzed, it is seen that the last column, in this case the top column, has the band for the protein, but also has light purple striations. Should the protein have been completely and perfectly purified, then there would only be the protein band. This means that the purification process was not complete and that there are still cell components other than protein in the second elution. A source of error could have resulted from capping the column too quickly during the flow through and not allowing all of the cell components not regarding the protein to pass through. Using the ladder as a molecular marker, the protein’s apparent molecular weight is about 25 kDa. In the sample 5 lane of the gel, there are no other bands of equal intensity to the protein; however, the estimated purity should not be 100% due to the fact that there are other lighter striations throughout the lane. This would bring the estimated purity to about 80%.

Conclusion: The purpose of these labs was to transform bacteria with DNA plasmids in order to allow it to adopt a certain protein and initiate replication. Following the production of the protein, the cells were lysed and the protein isolated in order to create a pure solution of the protein itself. The protein was then characterized and was given form on a gel in order to determine purity and estimated molecular weight. The experiment was successful in creating a protein solution that is about 80% purity. Possible future steps include using learned techniques in order to isolate a certain protein that has been worked with in PyMOL and or GOLD docking programs, in order to transfer digital theoretical work into the wet lab.

References:

Protein production and purification. //Nat Methods//. **2008,** //5,// 135-46.

Figure 1. Digital representation of the E. coli BL21 (DE3) with the plasmid DNA. It is shown here as grown on an LB agar plate with antibiotic to select for colonies that have the gene for antibiotic resistance. The purple tint of the bacterial colonies signal that the bacteria has "picked up" the plasmid DNA. Figure 2. Digital representation of the E. coli BL21 (DE3) without the plasmid DNA. It is shown here as grown on an LB agar plate with antibiotic; however, without the plasmid DNA, the bacteria lacks the gene for antibiotic resistance, and thus there is no visual indication that the bacteria is growing and thriving. Figure 3. Digital representation of the large culture made for overnight expression. The bacteria has been incubated in an LB and Ampicillin media overnight. Since DNA uptake in bacteria is not 100% efficient, it is important to get rid of the bacteria that did not obtain the Ampicillin resistance from the plasmid DNA; thus, the mixture with the Ampicillin/LB media left only the bacteria colonies that were transformed by the plasmid DNA and obtained that antibiotic resistance. Figure 4. Digital representation of the wet pellet made by centrifuging the bacterial solution in conical tubes. The LB/Amp media was decanted after the centrifugation so that all that remained was the wet pellet of bacteria cells. __Lab Protein Purification__ Figure 1. Digital representation of the Absorbance graph of the first elution made in the protein purification lab. The measurement was made at the wavelength of 280 nm. Figure 2. Digital representation of the Absorbance graph of the first elution made in the protein purification lab. The measurement was made at the wavelength of 574 nm. This wavelength was found to be the maximum wavelength for the protein, GBR22, used in this lab. Figure 3. Digital image of elution one made in the protein purification lab. The purple color shown represents the lesser purified protein of the two elutions. Six milliliters of solution were gathered in this elution. Figure 4. Digital image of elution 2 made in the protein purification lab. The clear color shown represents the most purified protein solution of the two elutions. Five milliliters of solution were gathered in this elution.