Title:
Analysis of pGEM-gbr22 through Protein Expression, Purification and Characterization

Introduction:

A plasmid is a circular piece of DNA that can code for a certain protein. A specific plasmid can be inserted into E. coli bacteria through a heat shocking method. A plasmid can then be used to transform competent E. coli bacteria in order to express a protein of interest for further analysis. E. coli bacteria was used because “it is fast and inexpensive to test a wide variety of possible strategies and one can complete a fairly comprehensive analysis within a relatively short period of time.” [1] The transformed bacteria can be grown in large quantities to overexpress a protein of interest and then the purification process can begin. Purification begins under certain “conditions [that] maximize cell lysis and the fraction of the recombinant protein that is extracted while minimizing protein oxidation, unwanted proteolysis and sample contamination with genomic DNA.”[1] Once the parts are separated the proteins can be placed in a column to further isolate a target protein in conjunction with a HIS-tag. The column and HIS-tag are pivotal parts of purification because “The final purity of the protein can be optimized by controlling the ratio of recombinant protein to the column size; lower-affinity contaminants can be competed with a relative excess of the histidine-tagged recombinant protein.”[1]. “The remaining proteins are separated on a porous matrix according to their size, thus not only contaminant protein with different size than the target protein but also different oligomeric forms of the same (apparently pure) protein can be separated.”[2] The porous matrix works through electrophoresis and eventually the size and purity of the target protein can be determined. The plasmid pGEM-gbr22 will be used to transform E. coli bacteria to overexpress a purple colored protein which will be purified through a column and a HIS-tag. The purified protein will then be run on a SDS-PAGE gel to determine the purity and length of the target protein. If the bacteria expresses the purple colored protein then through characterization the protein should have approximate size of 40 kDa.



Materials & Methods:

The pGEM-gbr22 plasmid was inserted into E. coli bacteria through heat shocking. The transformed bacteria were then placed on two agar plates, a plate that had ampicillin and a plate that lacked ampicillin that was used as a control to determine whether sterile techniques were used. The bacteria were left on the agar plates to grow overnight at 37C. The bacteria from the agar were placed into a starter culture and then into a larger liquid culture What kind of media was used? How long was the incubation period? to incubate and grow more in order to overexpress the purple protein. The bacteria from the culture were then centrifuged Include how long to create a pellet to isolate the cells from the liquid media. The pellet was suspended in a phosphate buffered saline solution and was frozen for a week. After a week, the pellet was thawed out then lysozyme and cyanase were added to break the cells down. Include how much of each (lysozyme, benzonase, etc. were added!) The broken down bacteria were then centrifuged to isolate the protein which is now in the supernatant. A small portion of the supernatant was taken as a sample. The rest of the supernatant was placed into a column that utilized a syringe filter and then a Ni-NTA was added into the column. The column now held the supernatant and Ni-NTA, a wash consisting of 1 mL 1x PBS was flowed through the column and taken as a waste sample. Then a flow consisting of 5 mL of 20 mM imidazole in 1x PBS was added to the column and a wash sample was taken. Two elutions were taken after the wash step using 5 mL of 250 mM imidazole in 1x PBS. Then samples of both elutions were taken. The samples that were taken were placed into a SDS-PAGE gel and electrophoresis was run. The gel was then stained using an imperial protein stain and then destained with water. The gel was then dried to further examine the proteins in the gel. The bands of the gel demonstrated the size and purity of the purple protein.

Mention company and location for PAGE ladder, spectrophotometer, etc.


Results:
VDSPEL1.jpg
Figure 1a: After 24 hours of incubation, the BL21(DE3) E. coli expresses the plasmid pGEM-gbr22 on the LB and ampicillin agar plate. The transformation of the E. coli is evident because of the purple color.
The BL21(DE3) took up the pGEM-gbr22 plasmid and expressed the purple colored protein. There is approximately 400 colonies on the agar plate which is more than expected due to the experimental errors which led to the addition of too much bacteria to the plate.


VDSPEL2.jpg
Figure 1b: After 24 hours of incubation, the BL21(DE3) did not grow on the LB and Ampicillin agar plate. There is no growth because the bacteria here were not given the pGEM-gbr22 plasmid.

There are no bacterial colonies on this agar plate because the BL21(DE3) E. coli did not have the pGEM-gbr22 plasmid which had ampicillin resistance. This was the control plate to determine sterile technique throughout the experiment.



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Figure 1c: After 24 hours of incubation, the fun plate did not grow any bacteria on a LB agar with no ampicillin.
There are no bacterial colonies on the fun plate. A kimwipe was wiped on the door handle and then dabbed onto the agar plate. The results indicate that there is little to no bacteria on the doorhandle. really???

VDSPEL4.jpg
Figure 2: After a 24 hour incubation, a large culture of the BL21(DE3) E. coli bacteria that expressed the pGEM-gbr22 plasmid was created.
A large culture was created from a colony that expressed the purple colored protein. The purple color indicates that the incubation worked properly and there is a large mass amount of E. coli bacteria that express the pGEM-gbr22 plasmid. RPM and temperature?


VDSPEL5.jpg
Figure 3: This pellet was isolated from the large culture by utilizing a centrifuge that spun at 5000 rpm for 10 minutes at 4C. The pellet weighed .39mg.
The large culture was placed into the centrifuge to isolate the bacteria. The pellet weight was determined to be .39mg.


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Figure 4: The elutions were created by using a 250mM imidazole and 1x PBS solution that was flowed through a column. Elution 1 is more purple than Elution 2 meaning that there is a higher concentration of purple protein present in Elution 1.
Through purification these two elutions were created. This demonstrates the purification process worked because elutions were purple indicating that there was purple colored protein present




ah1.jpg
Figure 5a: A nanodrop spectrometer was utilized at a wavelength of 280nm for elution 1 to determine to relationship between Wavelength and Absorbance.

ah2.jpg
Figure 5b: A nanodrop spectrometer was utilized again at a wavelength of 280nm for elution 1 to determine to relationship between Wavelength and Absorbance.
Elution 1 was placed into the nanodrop machine and the concentration was measured. This supports that there is protein present in the elution because the absorbance is greater than 0 and there is a peak at 280nm.

A=Ebc
Absorbance= .3125
Extinction coefficient = 38850 L/(mol cm)
Path length = 1 cm
.3125 = (38850 L/(mol cm))(1)(c)
c = 8.04 x 10^-6 mol/L

The concentration of elution 1 was determined to 8.04 x10^-6 mol/L.

Yield in mg? Please calculate the yield stated in the front page, not just the concentration.
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Figure 6: The samples collected from steps in the purification process were put into a 4-20% Tris-glycine gel (SDS gel) and then a gel electrophorisis was run to separate the proteins.

Gel electrophoris was used on this SDS gel and allowed the proteins to be separated based on size. This gel is helpful for determining the protein of interest's size and purity. Include what each lane contains in the sample.


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Figure 7: This is the Thermo-Scientific #26616 PageRuler Prestained Protein Ladder for a 4-20% Tris-glycine Gel or SDS gel.

This is a ladder that was run in the SDS gel to analyze the size of the purple protein. Mention company




Discussion:
The results of the SDS gel demonstrate that the purple protein was successfully expressed and purified. The lane that held Elution 1 had a band with high density which meant there was a high concentration of the purple protein. Since there is only one band that has high density that means that the purification process worked as expected. The experiment went successfully because there is a high amount of purple protein that was expressed and purified. There were many errors to this experiment that could have skewed the results. When actually pouring on the bacteria with the pGEM-gbr22, there was an error because there was too much of this bacteria added which led to too many colonies being formed. The mass amount of colonies could have effected the individual colony size which would have effected the amount of growth in the large culture and subsequently would give less purple protein then what was expected. Another error that could have skewed the data is that the nickel and resin did not have that much time to mix with the purple protein. This would lead to less purple protein being purified and would lead to a less dense band in the SDS gel than what would be expected.
Lysozyme was used in this experiment in order to break down the E. coli cell wall to expose the purple protein. Cyanase was used in the experiment to digest DNA/RNA in the E. coli in order to make the cell more viscous to make centrifugation more effective to isolate the protein. The HIS tag works because the purple protein has a chain of histadine that makes it different from the other E. coli proteins. The HIS tag has nickel and resin in it which is placed into the column with all of the proteins and the purple protein sticks to the nickel but the nickel is big enough so it wont go through the column. Imidazole is a chemical that competes with the histadine to attach to the nickel in the reisn. A weak amount of imidazole is first run through to wash away any other proteins. Then a strong amount of imidazole is run through the column to wash away the purple protein because the imidazole competes better and then the purple protein can be flowed through to create an elution. There were 6 samples that were used in the SDS gel. Sample 1 consisted of the E. coli that expressed the plasmid that was exposed to lysozyme. Sample 2 consisted of the supernatant after the cyanase was added and then centrifuged. Sample 3 is the flow of when water was flowed through the column to collect waste. Sample 4 is the flow through wash using 20 mM imidazole and 1x PBS. Sample 5 is elution 1 that used 250 mM imidazole and 1x PBS. Sample 6 is elution 2 that also used 250 mM imidazole and 1x PBS. The difference between the wash and elution buffer is that the wash had a 20 mM imidazole concentration and the elution had a 250 mM imidazole concentration. The size of the purple protein was determined to be 25 kDa. The purity of the sample was around 75% because there was another faint band in the SDS gel.
Conclusions:
The pGEM-gbr22 plasmid was inserted into E. coli bacteria in order for the bacteria to express a purple colored protein. The bacteria that expressed the purple colored protein were grown in a large culture and then purified. The purification process consisted of centrifuging and filtering through a column using a HIS tag. Samples from the purification process were placed into an SDS gel and electrophorisis was run to separate the proteins by size. Once the proteins were separated, the size and purity of the purple protein was able to be determined. The size of the purple protein is approximately 25 kDA and the purity was approximately 75%. Be careful with tenses in your writing! These results demonstrate that the HIS tag purification system work well and can be replicated again effectively. Expressing a protein and purifying it are important to the Virtual Drug Screening process because that protein can be a pivotal part of an infectious disease. Virtual Drug Screening can run simulations through computers to see which chemicals would best inhibit the protein. The chemicals can be added to the cells of the infectious disease and then expressed and purified (The chemicals, or ligands, aren't really expressed and purified- the proteins are. ) to see whether the chemical did anything to the concentration of the protein. If the concentration of the protein is lowered from this chemical, then that chemical could be a potential drug target.
References:
[1] Nat Methods. 2008 Feb;5(2):135-46. Protein production and purification.
[2] "Protein Expression and Purification Core Facility Protein Purification." Protein Expression and Purification Core Facility <http://www.embl.de/pepcore/pepcore_services/protein_purification/purification/index.html>. (Accessed April 16, 2013)

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