JohnN

=The Protein Expression, Purification and characterization of GBR-22.=

=Intro:= Given a sample protein of interest to study, it is necessary to have sufficient amounts of the protein to study from. Hence, in the first part of the three-part lab series, the primary objective is to over-express the protein. This part is taken care of thanks to E. Coli's ability to integrate proteins of different species and express recombinant protein. E. Coli is of ideal choice when it comes to protein expression because it is able to take in genetic information from other species, combine it with E. Coli's own genetic information through a process called genetic recombination and express it. What makes E. Coli also ideal is the simplicity and ease during its duplication. Given a simple nutrient source such as agar and a simple place to grow such as a petri dish, E. Coli can grow exponential-like. Other organisms may not be able to do the same, due to the time necessary for reproduction (such as dogs), moral conflicts (such as forcing humans to take in potentially harmful material), and the mixing of genetic information from sexual reproduction. =Materials:= Petri dishes, Sample Protein (GBR-22), Agar, Ampicillin, conical tubes, round bottom tubes, centrifuge, centrifuge tubes, ring stand with clamps, Ni-NTA resin, Benzonase, Imidazole, TGS, PBS of various concentrations. Incubating-fridge, -20 degree centigrade fridge, hot water bath, SOC media, sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE), burner, shaking incubator, LB media, Ice bucket, nanopure water, deionized water, various micropipettors with various tips, pipettes of various capacities, nanodrop spectrophotometer (Thermo Scientic, Wilmington, DE). =Methods:= Given a sample protein of interest to study, it is necessary to have sufficient amounts of the protein to study from. Hence, in the first part of the three-part lab series, the primary objective is to over-express the protein. By using E. Coli's ability to integrate proteins of different species and express recombinant protein, it makes the over-expression of protein possible. In the second part of the lab series, the protein was separated from other materials such as cell debris from E. Coli that is not of interest by lysing the cell and running the solution through a centrifuge. After separating the soluble protein from the insoluble-cell debris the protein was purified through a series of washes and rinses using various concentrations of immidazole and Ni-NTA resin to bind to the protein. Ni-NTA is able to hold the protein back from the low concentration immidazole washes because it competes with the 6 Histidine tail of GBR-22. When the concentration of immidazole is high, both the Ni-NTA resin and the protein passes through the filter. Lastly, to study the protein, in this case, gel electrophoresis was used to analyze purified protein samples. =Results:= Figure 1 - LB Agar Plate with E.Coli bacteria grown with AMP present. Amp resistant bacteria has a purple florescence. Condensation present. Numerous colonies! Near impossible to count by band.

Figure 2 - LB Agar Plate with E.Coli bacteria grown with AMP present. Bacteria did not survive the antibiotic: AMP. Condensation present. No colonies present.

Figure 3 - FUN Plate with fish tank bacteria grown. No AMP present in the plate. Bacteria colonies shown in yellowish little circles.

Figure 4 - Flask #1 with protein that was grown in LB and AMP.

Figure 5 - Flask #2 with protein that was grown in LB and AMP.

Figure 6 - Conical #1 with wet protein pellet after being centrifuged and liquid removed.

Figure 7 - Conical #2 with wet protein pellet after being centrifuged and liquid removed.

=Protein Purification Lab=
 * [[image:Elutions.jpg width="240" height="316" caption="Figure 8: Elution 1 on the left appears purple suggesting that it contains the isolated gbr22 protein. The protein was purified or removed from solution by using Ni-NTA with 250mM Imidazole. Elution 2 appears clear suggesting the absence of gbr22. This is likely due to the apparent success in Elution 1 where most of the protein was purified."]] ||


 * [[image:jnlcpElution280run1.jpg width="709" height="380" caption="Figure 9: The absorption spectrum of Elution 1 from the first trial using the Nanodrop spectrophotometer. The absorbance at the 280 nm wavelength was 0.043."]] ||


 * [[image:jnlcpElution280run2.jpg width="710" height="442" caption="Figure 10: The absorption spectrum of Elution 1 from the second trial using the Nanodrop spectrophotometer. The absorbance at 280 nm wavelength was 0.09. "]] ||


 * [[image:jnlcpElution574run1.jpg width="709" height="378" caption="Figure 11: Based on the absorption spectrum of Elution 1's first trial: the maximum absorption wavelength was 574 nm. The absorbance at 574 nm is 0.20 because we multipled 0.02 by 10 to accommodate for the 1 mm path length of the spectrophotometer."]] ||


 * [[image:jnlcpElution574run2.jpg width="710" height="435" caption="Figure 12: Based on the absorption spectrum of Elution 1's first trial: the maximum absorption wavelength was 574 nm. The absorbance at 574 nm is 0.23 because we multiplied 0.023 by 10 to accommodate for the 1 mm path length of the spectrophotometer."]] ||

=Protein Characterization Lab=





Beer's Law Calculations: A=εbc MW of gbr22 = 25794.2 Extinction coefficient at 280 nm = 38,850 The absorbance at 280 nm wavelength was 0.09. 0.09 = (38,850) (1)C C= 0.09/38,850 Yield at 280 nm: 9.7(C) = 2.247 x 10^-5 moles per liter (2.247 x 10^-5) x (25794.2) = 0.580 grams

Extinction coefficient at max (574 nm) = 118,300 The absorbance at 280 nm wavelength was 0.23. 0.23 = (118,300) (1)C C= 0.23/118,300 Yield at 574 nm: 9.7 (C) = 1.886 x 10^-5 moles per liter (1.886 x 10^-5) x (25794.2) = 0.486 grams

=Discussion:= From the results obtained from observing the Agar control plates with the DNA plate, it supports that the plates were likely not to be contaminated. Even though the lab is contaminated as the results of the fun plate indicates, the use of the burner prevented contamination. Ampicillin was used to determine help prevent E. Coli without the recombinant DNA from proliferation. The purification of the targeted protein appears to be successful as seen in Figure 8: where in Elution 1 appears purple suggesting that it contains the isolated gbr22 protein. Elution 2 appears clear suggesting the absence of gbr22. Elution 1 ideally where most of the protein should have been harvested to suggest successful purification. This was verified in the (SDS-PAGE) where the last two lanes were from samples of Elution one and two respectively. The faint mark in Elution two supports the estimated 99% pure protein in Elution 1. Also, the actual molecular weight of the protein is known to be 25.79 kiloDaltons. By analyzing the SDS-PAGE and comparing the protein ladder as a reference reference to elution 1, it can be estimated that the purified protein's molecular mass is 25 kiloDaltons, hence the protein appears to besuccessfully purified.On the other hand, the numbers obtained using the nanodrop spectrophotometer appears low in Figures 9 through 12. =Conclusion:= From the results obtained the purification of the targeted protein appears to be successful. In the beginning, by observing the Agar control plates with the DNA plate, all the way until the end with the SDS-PAGE gels, there are results to supports that the protein was successfully inserted as recombinant DNA, selected, duplicated and purified with an estimated 99% pure protein in Elution 1. The low numbers obtained using the nanodrop spectrophotometer seen in Figures 9 through 12 remains a concern as a precautionary warning that there could possibly be errors that occurred in the lab or that it could be a technical error when using the spectrophotometer. Other possible sources of error include incubation times that were over the specified limit and centrifugation in an instance where temperature control was not available. =References:= Fermentas Molecular Biological Tools. PageRuler Prestained Protein Ladder. [] (accessed April 18, 2011).