EstherM

=Protein Discovery; Lab Report=


 * Introduction**

Protein expression, purification, and characterization can be used to determine the weight of a certain protein, and the purity and yield of a final purified protein product, in this case gbr22 protein. Proteins are purified for crystallography or enzyme inhibition assays and therefore need to be oxerexpressed if there is a low number of protein in the native organism. The plasmid used in this lab is pGEM-gbr22 and contains the coding for a fluorescent protein that makes if easy to follow the expression and purification process since it is purple and can be detected by absorbance. Protein expression can vary depending on the approach used to optimize the expression. For example, time or temperature of the induction and the concentration of the inducer can affect how much is expressed. Also, when the mRNA is not as stable, this slows down translation. Other factors involving mRNA include premature termination of transcription or translation, mutations within the sequence, or inhibition of protein synthesis and cell growth. As a result, expression is low; however, this can be improved by replacing codons that are not often found in a highly expressed gene with codons that are occur more often throughout the gene. In the process of protein purification, to optimize the purity of a protein, a second elution process is completed, often either an ion-exchange chromatography, hydrophobic interaction chromatography, or gel filtration chromatography. In research, proteins are extracted to determine their three-dimensional structures, accomplished by using protein crystallography to understand gene structure. Purified protein was crystallized to form diffraction-quality crystal. Thousands of protein crystallization conditions can be screened with today’s scientific and technological advances, though it is still a work in process. Structural genomics involves understanding the three-dimensional protein structure corresponding to the coding DNA, and comes from a structural library of different proteins from different organisms. In other areas of research, recombinant proteins are used that also go through the process of protein expression, purification, and characterization. Questions that arise when working with this include how the protein should be expressed, whether in bacteria, yeast, insect cells, or human cells. These problems can be difficult to answer at times considering that every protein is different and there is no particularly correct answer that can be determined without extensive research. Various methods can be used and applied to large numbers of proteins. Protein characterization of purified protein reduces the possibility of wasting resources on protein material of low quality. Characterization demonstrates that different samples of the same protein can have similar properties. In this lab, protein was expressed and purified, then characterized using gel el ectrophoresis andSodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to estimate the molecular weight of the gbr22 protein as well as the purity and yield of the final purified protein product.


 * Materials & Methods**

The first step of the procedure is Protein Expression. This is the process of overexpressing a recombinant protein in bacteria by transforming competent bacterial cells with DNA plasmid. A starter culture of bacteria was grown overnight, and this culture was used to inoculate a larger culture and express a recombinant protein. This lab required at least three days. There were two lab plates, both with antibiotic, and one with DNA and one was a control. These cells were harvested then put in the freezer for the next lab. The second lab involved protein purification of the overexpressed bacteria from t he previous lab. This lab involved breaking open bacterial cells to release the soluble protein inside. The insoluble cell debris were removed (including the cell wall, membranes, and inclusion bodies) through centrifugation. The proteins were purified using the affinity tag and Ni-NTA resin. Different samples were collected to be analyzed in the next lab, protein characterization. In this lab, gel electrophoresis was performed to analyze the protein samples that were collected during the expression and purification labs. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate proteins in the samples. Using the electrophoresis and spectroscopy results, the molecular weight of the gbr22 protein as well as the purity and yield of the final purified protein product were calculated. This data was then correlated to the UV-Vis spectroscopy measurements that were made in the last lab to estimate the concentration of the protein solution.

= = = Lab Protein Expression =
 * Results**

Fig. 1 Ampicilin plate with plasmid DNA, after one day of cultivation; bacterial growth in colonies, approximately hundreds of colonies

Fig 2. Control Ampicillin plate with no DNA added; Fun plate Figure 3: LB plate without antibiotics growing bacteria taken from the fish tank. Figure 4: Cell pellet with purple colony after centrifugation, 0.39 g

Figure 5. Elution 1 and 2 from Protein Purification lab Figure 6. Nanodrop spectophotometry Trial 1, spectrophotometer reading at absorbance 280

Figure 7. Nanodrop spectophotometry Trial 2, spectrophotometer reading at absorbance 280 Figure 8. UV/Vis spectophotometry Trial 1 Figure 9. UV/Vis spectophotometry Trial 2 Figure 10. Gel electrophoresis run FIgure 11. Protein Ladder molecular weight marker

Maximum absorbance wavelength - 574 λ Extinction coefficient at max λ - 118300 Absorbance at 280 = 0.64 Absorbance at 574 = 1.13 Extinction Coefficient at 280 nm = 38850 Molecular weight of gbr22 = 25794.2 A = Ebc 0.64 = (38850)(1)C C = 1.637x10^-6 Purified protein collected = 9.7 mL Yield = 9.7 Yield using 280 wavelength = 9.7(1.647)(10^-5) = 1.5976x10^-4 mg Yield using max. wavelength = 9.7(9.552)(10^-6) = 9.2643x10^-5 mg


 * Discussion**

The concentration of the protein was calculated to be 1.637x10^-6 mg/mL. this did not exactly match the concentration found at an absorbance of 280 nm, but it was very close, showing that our data was relatively accurate. In the DNA gel electrophoresis, the 2nd column was the protein ladder, which served as a reference for judgint the size of the other bars. Judging from the Molecular weight standard, the estimated molecular weight of the purified protein is 100 dKa. the 3rd column was the cell lysate, the 4th column was the solute and pellet, the fifth was flow thrgouh, in the sixth was wash buffer, the 7th was Elution 1, and the eight was Elution 2. the cell lysate, the solute and pellet, and the flow through contained more of the unfiltered protein. In the sample 5 lane, there are four clearly marked bands of protein, but one band was the most prominent, showing that the resutls are somewhat pure. The estimated purity is very close to 100% due to the fat that the one band is very clear, and also is almost the only band present on the sample 6 lane.


 * Conclusion**

In this lab, protein from a recombinant DNA in bacteria was overexpressed then purified.Gel electrophoresis was then used to analyze the different samples collected of protein at different states of the experiment. From there, it was possible to calculate the molecular weight of the gbr22 protein, and the purity and the yield of the final purified protein product. From this, it is possible to continue further research and discover more ways to optimize the expression of protein, to purify the protein, and other ways to analyze the data. 
 * References**

**Chayen, N.; Saridakis, E. **Protein crystallization: from purified protein to diffraction-quality crystal. **Nat Methods. ** **2008 ** **Feb;5(2):135-46. **   **European Molecular Biology Laboratory. Protein Expression and Purification Core Facility ** Protein Purification (acessed April 18, 2011) 