GRANT+T

Reviewed by Larry H. Overall Great Job! __ Title: __ Exploring the Purification, Expression, and Characterization of pGEM-gbr22 __ Introduction: __ The bacteria, Escherichia coli, particularly the BL21 (DE3) strain, are is commonly used throughout research as an expression host for the production of recombinant proteins because of the high soluble protein yields [1]. This method is very popular among the biological and biomedical sciences [1]. The production of recombinant proteins can be broken down into three steps: Protein Expression, Protein Purification, and Protein Characterization. A common protein expression method is to pick a single colony from a streaked plate of host containing the recombinant vector and growing a starter culture using an appropriate anti-biotic [2]. Protein Purification is a method of filtering the desired protein from any unwanted protein or cell matter. A C-terminal hexahistidine affinity tag is used to specifically select the protein of choice through a filtering process as long as the protein is mostly soluble [1]. This affinity tag will bind to the column and all not all of the untagged proteins will flow through, some will get stuck since some proteins will randomly have stretches of His repeats of the untagged proteins will filter away leaving the desired protein in the column not just proteins get filtered out in this process. Protein Characterization is the last step. Characterizing the purified protein minimizes wasting time and resources on poor, inadequate proteins and it insures the protein is of good quality [1]. SDS-Polyacrylamide Gel Electrophoresis is used to denature the protein and check the purity of the sample expand on how SDS-PAGE works. The stained gel bands will demonstrate a proportional amount of protein based on the intensity of the band [1] vice-versa: the intensity depends on the amount of protein. If the desired protein was expressed properly, the band of intensity should correlate to the molecular weight on the protein. In this lab, the protein, pGEM gbr-22 pGEM is a plasmid, not a protein MW 25 kDa, was expressed, purified, and characterized. If the production of the protein is sufficient, the SDS-PAGE gel will show a single band of intensity around 25 kDa. __ Materials & Methods: __

The host cell, //E. Coli// BL21 (New England BioLabs, Ipswich,MA), which was ampicillin resistant, was transformed with an expression plasmid pGEM-gbr22. First, the bacteria were added to test tubes and heat shocked. Then, SOC media was added to prevent contamination and samples were placed on agar plates to incubate overnight specify the actual # of hours at 37⁰C. After the cells grew, a culture was selected and placed in a flask with nutrient rich LB and incubated. The protein was then harvested using the Allegra X-15 Centrifuge (Beckman Coulter,Inc. Brea, CA). 1x PBS and lysozyme were added to the purple harvested pellet and stored at -20 ⁰C. Later, Benzonase was added to the conical tube with the lysate and centrifuged. A syringe filter was used to remove large particulate matter from the lysate. Now, Ni-NTA resin/buffer company info? was mixed with the protein and a homogenous solution was created. A Bio-Rad chromatography Econo column and a buffer with high imidazole concentration were then used to isolate the protein. The protein was now in its purest form. A Nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE) was used to depict the concentration of the protein. An Electrophoresis module was assembled. A 1x TGS buffer was used to fill the tank. The samples were then loaded into the 10 wells. The Electrophoresis module then produced a gel. Lastly, the gel was stained and dried. Separate your M&M into sections for better readability

__ Results: __ Explain exactly what positive control means.

Mention the pinkness and what it means Mention the purpleness Mention the lack of purpleness Pellet of what?

Beer's Law: A = Ebc

Molecular weight of gbr-22 = 25,794.2 g/mol


 * 280 nm **

A = 0.186, E = 38,850 L/mol*cm, b = 1 cm

c = A/Eb

= (0.186)/(38350 L/mol*cm)(1 cm)

= (4.85 x 10-6 mol/L)(25794.2 g/mol)= **__0.125 mg/ml concentration__**

Yield = cV

= (5ml)(0.125 mg/ml) = __**0.625 mg yield**__


 * 574 nm **

Average absorbance of 2 trials: (0.32+ 0.23)/2 = 0.055

A = 0.055, E = 118300 L/mol*cm, b = 1 cm

c = A/Eb

= (0.055)/(118300 L/mol*cm)(1 cm)

= (4.9 x 10-8 mol/L)(25794.2 g/mol) = **__0.00126 mg/ml concentration__**

Yield = cV

= (5ml)(0.00126 mg/ml) = **__0.0063 mg yield__** Label your lanes like this: Lane 1: skip Lane 2: ladder Lane 3: cell lysate etc... __ Discussion: __

At 280nm the protein concentration was calculated to be 0.125 mg/ml and at 574nm the protein concentration was 0.00126 mg/ml. The protein yield at 280 nm was 0.625 mg and 0.0063 mg at 575nm. Based on Figure 12, the protein was in fact expressed as seen by the intense band at about 25kDa. However this band is not the only band present in elution 5. There is another intense band present at about 18 kDa resulting in the conclusion that the protein was about 50-60% pure. The cause of error would have been contamination throughout various steps of the expression process or poor sterile techniques. It was probably the purification column that was at fault

Throughout the purification process, many different steps were taken to isolate the desired protein. Two key enzymes, lysozyme and Cyanase were used to isolate the protein. The Lysozyme was used to break down the cell wall of the bacteria and Cyanase was used to remove excess nucleic acid from the protein solution cyanase degrades, not removes. The C-terminal hexahistidine affinity tag allowed the gbr-22 to bind to the Ni-NTA. This binding allowed unwanted proteins to be washed out of the column using a wash buffer with a small amount of imidazole. An elution buffer with a high concentration of imidazole was then used to release the tagged protein and capture the pure gbr-22 Why do we use imidazole for both wash and elution? . Throughout this process, 6 samples were taken after key steps. Sample 1 was taken from the LB and ampicillin mixture, Sample 2 was obtained after syringe filtering the media, Sample 3 was obtained after the buffer/resin mix was washed through the column, Sample 4 was obtained after the 20mM imidazole and 1x PBS wash step, Sample 5 contained the gbr-22 after the Elution 1: 250mM imidazole and 1x-PBS was poured through the column, and lastly, Sample 6 was taken after the last elution buffer was poured through the column to catch an lingering protein in the column. This sample information should be in the caption of the image. You should analyze the results of your gel in the discussion.

__ Conclusions: __ In this lab, the purple coral protein was expressed in the E. Coli BL21 (DE3) bacteria, purified using lysozyme lysozyme is not used to purify the sample and the HIS tag system in combination with the Ni-NTA resin to filter away excess cell debris and protein, and lastly characterized with the SDS-PAGE analysis. We concluded that our protein was expressed, but the purity was around 50-60% because of contamination. These techniques will be used in the study of protein targets for drug discovery. Be more specific for future directions, i.e. assays, crystallography, etc...

__ References: __

[1]  Gräslund, S.; Nordlund, P.; Weigelt, J.; Hallberg, B. M.; Bray, J.; Gileadi, O.; Knapp, S.; Oppermann, U.; Arrowsmith, C.; Hui, R.; Ming, J.; dhe-Paganon, S.; Park, H. W.; Savchenko, A.; Yee, A.; Edwards, A.; Vincentelli, R.; Cambillau, C.; Kim, R.; Kim, S. H.; Rao, Z.; Shi, Y.; Terwilliger, T. C.; Kim, C. Y.; Hung, L. W.; Waldo, G. S.; Peleg, Y.; Albeck, S.; Unger, T.; Dym, O.; Prilusky, J.; Sussman, J. L.; Stevens, R. C.; Lesley, S. A.; Wilson, I. A.; Joachimiak, A.; Collart, F.; Dementieva, I.; Donnelly, M. I.; Eschenfeldt, W. H.; Kim, Y.; Stols, L.; Wu, R.; Zhou, M.; Burley, S. K.; Emtage, J. S.; Sauder, J. M.; Thompson, D.; Bain, K.; Luz, J.; Gheyi, T.; Zhang, F.; Atwell, S.; Almo, S. C.; Bonanno, J. B.; Fiser, A.; Swaminathan, S.; Studier, F. W.; Chance, M. R.; Sali, A.; Acton, T. B.; Xiao, R.; Zhao, L.; Ma, L. C.; Hunt, J. F.; Tong, L.; Cunningham, K.; Inouye, M.; Anderson, S.; Janjua, H.; Shastry, R.; Ho, C. K.; Wang, D.; Wang, H.; Jiang, M.; Montelione, G. T.; Stuart, D. I.; Owens, R. J.; Daenke, S.; Schütz, A.; Heinemann, U.; Yokoyama, S.; Büssow, K.; Gunsalus, K. C.; Consortium, S. G.; Consortium, C. S. G.; Consortium, N. S. G., Protein production and purification. Nat Methods 2008, 5 (2), 135-46.

[2] Protein Expression and Purification Core Facility. [] (accessed April 17).