RonnieS

=__Protein Production and Purification__= = =

Characterization and Purification of Overexpressed Fluorescent pGEM-gbr22 Protein Transformed to //E.coli// B21

 * __Introduction__**

The chemical synthesis of protein using expression, purification, and characterization are used for many applications involved in the biological and biomedical sciences that yield a highly pure targeted protein. Our protein of interest in lab was gbr22 bacteria. More specifically, we selectively targeted the plasmid (pGEM-gbr22) vector that encodes for fluorescent and/or colored proteins, and polynucleotide sequences cloned from its native coral of the Great Barrier Reef. The fluorescent and polynucleotide proteins serve as a marker or detectable labels in tracing the experimental progress during expression and purification. A widely used expression system is the //Escherichia coli// bacteria host, which allowed the (pGEM-gbr22) vector to become transformed into the //E.coli// BL21 host. The //E.coli// B21 host possesses an ampicillin resistant gene and a fluorescent gene with six histidine residues attached to the C-terminus.

Protein purification is vital for the characterization of proteins and involves multiple steps in isolating the protein of interest, gbr22. The main mechanism employed is the separation of protein from the matrix and then isolating the desired protein from the other protein. Furthermore, the hex-histidine tag of (//E.coli// BL21(DE) pGEM-gbr22) of the overexpressed bacteria has an affinity towards nickel ions on the Ni-NTA resin. By immobilizing the nickel ions on the Ni-NTA resin, the six histidine residues bind to the Ni-NTA resin, leaving the histidine-tagged protein behind the chromatography column. This process creates a fast and efficient protein purification since the nonspecific matrix components will flow through the chromatography column. Usually following step, the targeted protein is released from the chromatography column, called elution, as a result of adding imidazole. The addition of imidazole competes with the histidine residues for the binding of nickel ions on the Ni-NTA resin. A second elution is run with a stronger concentration of imidazole to ensure that no contaminants are left.

Protein characterization quantitatively assesses the previous protein preparation methods and monitors the efficacy of each protein separation step. The procedure followed in our lab was sodium dodecyl sulfate polyacrylamide gel electrophoresis that separates proteins in a porous gel using an electrical current. The sodium dodecyl sulfate denatures the protein and gives it a negative charge proportional to the protein mass. Thus, the negatively charged proteins will migrate across the gel towards the anode side. The mass of the protein can be calculated in comparison to the molecular weight standards, and the concentration of the proteins can be measured with spectroscopy.


 * __Materials & Methods__**

There was an experimental plate with DNA and a control plate without DNA used during the transformation of competent bacterial cells. 25 m l of pGEM-gbr22 was added to each of the transformation tubes (experimental and control). The plasmid DNA was spun using a mini-centrifuge, and 2 m l of plasmid was added to the experimental tube. After 30 minutes on ice, the tubes were heat shocked in 42 oC water bath for 45 seconds. The tubes were placed on ice for 2 minutes, and 200 ul of SOC media was added. The tubes were shaken in an incubator for 30 min at 37 oC at about 250 rpm. Then, 5 colirollers and 50ul of bacteria/SOC mixture from tubes were placed into each plate. The plates were shook for 2 minutes and covered with lid. The plates were inverted and stored in 37oC incubator overnight. Ampicillin was added to each tube of 5 ml of LB in a sterile culture tube with cap. A single colony of (//E.coli// BL21(DE) pGEM-gbr22) growing on the LB/agar plate was swabbed with a sterile pipette tip and transferred to LB/amp media. The LB/amp media was placed into a rack in the shaking incubator and grown during the day for approximately 8 hrs at 37 C and 200-350 rpm. The plates were wrapped in parafilm and stored in the 4OC fridge. 25 ml of fresh LB was transferred to two 125 ml Erlenmeyer flasks. 50 m l of ampicillin was added to the LB to make the final concentration 100 m g/ml. Then, 0.625 ml of the starter culture was transferred from the tube to the 125 ml flasks. The flask was placed in a shaking incubator at 37oC and 350 rpm to grow for approximately 16-24 hrs. Once the media was purple, a 500 ul sample of the culture was taken and dispensed into a labeled eppendorf tube. The bacteria was poured into a 50ml conical tube. Next, the tubes were spun for 10 minutes at 5,000 rpm and 4OC. The purple pellet at the bottom of the tubes was weighed. Then, the cells were resuspended in a 1x PBS solution, and 50 ug/ul of lyzozymes were added to a final concentration of 1 mg/ml and vortex again. Then, the lysosome (2.5 ml suspension) was added to digest the //E.coli// cell walls. The solution was incubated for 20 min at room temperature and mixed occasionally. 2 m l of Benzonase was added to the 50 ml conical and mixed well by inverting the tube several times. The conical was incubated for 15 min at room temperature. The lysate was distributed into several 1.7 ml microcentrifuge tubes and centrifuge for 20 min at 14,000 rpm at 4 ° C. 50 m l sample of the supernatant was dispensed into a microcentrifuge tube and labeled as sample 2.

The liquid supernatant from the tubes was pipetted into a clean 15 ml conical tube leaving the cell debris pellets behind. Then, the lysate was ran through a syringe filter into a 14 ml round bottom transformation tube. A 50 m l sample of the flow through was taken as sample 3. The Ni-NTA resin was washed with 5 ml of 20 mM imidazole in 1x PBS and then a second wash. A 50 m l sample of the flow through was taken as sample 4. Then, 5 ml of the buffer containing 250 mM imidazole was added, and the buffer was collected in a 15 ml conical tube (Elution 1/sample 5). This was repeated by adding 5 ml of elution buffer and collecting it in a 15 ml conical tube (Elution 2/sample 6). Next, UV-Vis spectroscopy was used to measure the concentration of the final purified protein. 8.33 m l of 6x loading buffer was added to samples 2-6 and pipetted to mix. The samples were heat blocked at 95 ° C for 5 min. Then, the samples were centrifuged for 2 min at 5,000 rpm. 7 m l of the MW standards (PageRuler Prestained Protein Ladder #SM0671) were loaded into the second well. 20 m l of samples 3-6 was loaded respectively into wells 3-7. The gel electrophoresis was ran at 200 V for 25 min. After this was done, the gel was removed and washed three times for five min each with nanopure water. Then, the Imperial protein stain (30 ml) was mixed and placed on the orbital for 1.5 hrs. Next, the gel was rinsed twice with nanopure water, and its excess stain was soaked up with a KimWipe. The gel was left on the orbital shaker overnight. The gel was removed from the dish and dried at 75OC on Gradient cycle for 1.5 hrs.

**Lab Protein Expression**






** Lab Protein Purification **
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Molecular Weight = 25794.2 g/mol Absorbance = 0.240 Extinction Coefficient = 38850 L/mol-1cm-1

Beer's Law Concentration: C = A/Eb = [(0.240)/ (38850 L/mol-1cm-1)(1cm)] = 6.18 x 10-6 mol/L

Determining the concentration (mg/ml) at the wavelength 280 nm: C = (6.18 x 10-6 mol/L)(1L/1000ml)(25794.2 g/mol)(1000mg/1g) = 0.159 mg/ml

Percent Error of Concentration: [(0.240-0.159mg/ml)/0.159](100) = 50.94%

280nm Wavelength Yield: (0.24mg/ml)(5ml) = 1.2mg

Maximal Wavelength Yield: 574

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**Lab Protein Characterization ** ======





__**Discussion**__

Figure 1 represents the overexpressed bacteria differentiated by the experimental and control plates. The //E.coli// BL21 competent cells allowed us to conjugate the uptake of plasmid bacteria into the host. As a result, we were able to determine the efficacy of protein expression by the ampicillin resistant gene of (//E.coli// BL21(DE) pGEM-gbr22) indicated as purple. The control plate without ampicillin were conclusive to the experiment; the purple fluorescent expression was not spontaneous. The purple and transparent colored elutions in figure 4 indicated the level of protein purity. Elution 1 was obtained by the first imidazole wash, which means that there is a possibility of foreign material in the gbr22 solution. Elution 2 is transparent in color due to the heavy concentration imidazole wash, which is theoretically more pure than elution 1. During the first UV-VIS mode Nanodrop Spectrophotometry (Thermo Scientific, Wilmington, DE), we obtained a negative absorbance measurement. Therefore, we had to conduct a second spectrophotometry reading seen in figure 6 with another group’s elution buffer. The absorbance using the different elution buffer at 280 nm resulted in a percent error of 50.94%. During the SDS gel electrophoresis, we had forgotten to take the original gbr22 samples and mix with staining so we had to start the experiment over. In figure 8, samples 2-4 are represented by several dark bands along the length of the wells. This was most likely due to the high significance of other proteins in the matrix that was allowed to flow through during Ni-NTA resin purification. Sample 5 have fewer bands and are marked with a lighter color. Sample 6 has only one band, which signified that we have only gbr22 protein. Therefore, the imidazole outcompeted the histidine residues for nickel ion binding.


 * __Conclusion__**

Ultimately, the gbr22 bacteria was overexpressed and cultured. The //E.coli// cells were lysed with Benzonase and centrifuged to separate the soluble proteins. Then, the recombinant protein was purified using an affinity tag and Ni-NTA resin to bind the protein to the chromatography column. By adding imidazole, we were able to obtain elutions of our pure protein that we later used for protein characterization. The absorbance and concentration of the elution 1 was found using Nanodrop Spectrophotometer (Thermo Scientific, Wilmington, DE). The SDS gel electrophoresis separated the proteins and determined the purity and yield of the protein. Based upon the experiment, one could determine the threshold values for protein purity, protein production, and discuss further optimization of the target protein in the future for wet lab assays.


 * __Bibliography__**

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