Michael+T

__ Title __

The Expression, Purification, and Characterization of pGEM-gbr22 Using //E. Coli// BL21
__Introduction:__

// E. Coli // is lacks ion and ompT proteases and is compatible with a T7 lacO promoter system making it an advantageous host for protein production [1]. Using an //E. Coli// system allows for a method that is cost effective and efficient; the bacterium is commonly as an expression host for higher-level organisms. In order to express a protein, the a recombinant protein plasmid needs to be genetically modified to resist ampicillin. It would then be inserted into the //E. Coli// through heat shocking. A recombinant plasmid with resistance to ampicillin is necessary to kill off the //E. Coli// bacteria that did not express the desired protein, when placed on an ampicillin agar plate. The purification process overall is quite difficult. Every protein is different and there are various different protocols for purification [1]. For the purposes of this lab, once the protein had been cultivated, the bacteria were lysed in order to clarify the protein. The purification process involves running the protein through a column that contains a column matrix such as nitriloacetate agarose (Ni-NTA) [2]. The protein needs to have an affinity for the matrix used in the column. The recombinant protein in this lab contained six histidine residues, which allowed for separation. These residues binded to divalent cations. The protein can then be released from the Ni-NTA matrix through the addition of imidazole, which competed with histidine to bind to metal such as nickel.

__ Materials & Methods: __

pGEM-gbr22 was first expressed in //E. Coli//. //E. Coli//BL21 (DE3) was placed in two different transformation tubes. pGEM-gbr22 plasmid was added to one of the tubes and the two tubes were then heat shocked. SOC media was then added to each of the tubes and placed in a water bath incubator (250 RPM) for thirty minutes. The two separate samples were then added to agar plates containing ampicillin, and incubated overnight. A single colony of each was placed in two conical tubes each containing LB and ampicillin. The two tubes were then incubated for eight hours (37 degrees Celsius). 0.625 ml of the culture containing pGEM-gbr22 was pipetted into an Erlenmeyer flask filled LB and ampicillin. The flask was then placed in the water bath shaker for twenty-four hours. The contents of the flask was poured into a 50 ml conical tube and placed into an Allegra X-15 benchtop centrifuge (Beckman Coulter, Inc., Brea, CA). The supernatant was removed, and the pellet was mixed with 2.5 ml of 1x PBS solution. Lysozyme was then added to the tube and stored (-20 degrees Celsius). Afterwards, the pGEM-gbr22 was purified. Two microliters of Benzonase (Sigma-Aldrich, St. Louis, MO) was added. The contents of the tube was distributed amongst three Eppendorf tubes and centrifuged. The supernatant from all three tubes were then placed in a conical tube and filtered through a PES syringe filter (Membrane Solutions, Plano, TX). An Econo column (Bio-Rad, Hercules, CA) was used to run the supernatant with Ni-NTA resin/buffer through. Five ml of wash was then run through the column, followed by 5 ml of Elution, and then another 5 ml of Elution. A Nanodrop spectrophotometer (Thermo Scientific, Wilmington, ED) was used to measure the absorbance of the protein from Elution 1 at 280 nanometers and 574 nanometers. Finally the protein was characterized. The samples were prepared using a 6x gel-loading buffer. All the tubes were then placed on a heat block for five minutes and centrifuged at 5,000 RPM. An SDS electrophoresis was run for twenty-five minutes at two hundred volts. The first well contained the protein ladder (Thermo Fisher Scientific, Waltham, MA) #SM0671, the following ladders contained samples one through six, and four through six from a partner. The gel was then stained. The next day the gel was placed on Whatman filter paper (GE Healthcare, Maidstone, UK) and covered with cellophane. The gel was then dried.

__ Results: __



__ Discussion: __

All of the bacteria not containing pGEM-gbr22 were killed off when placed in the plate treated with ampicillin. One colony from the plate was placed in a tube containing LB and ampicillin and incubated and expressed. From this, Sample 1 was collected, and is comprised of //E. Coli// that contains pGEM-gbr22, LB, and ampicillin. An Allegra X-15 centrifuge was then used to spin down the bacteria, followed by the removal of the supernatant. Lysozyme was added in order to obliterate the bacterial cell wall of //E. Coli//. This essentially killed off the bacteria. Benzonase (a nuclease) was added and reduce the viscosity of the mixture by digesting the DNA and RNA to make the purification process easier. In order to remove cellular debris (waste that was not pGEM-gbr22), the mixture was centrifuged, leaving insoluble debris in a pellet. Sample 2 was taken from the supernatant, and therefore contained only soluble proteins.

The supernatant was then filtered, followed by the addition of the Ni-NTA resin. Ni-NTA has an affinity for histidine residues, and as a result attracted pGEM-gbr22. This tagging system essentially targeted the six histidine structures found at the C terminus of pGEM-gbr22. The proteins that Ni-NTA did not have a binding affinity for were removed when placed in the column. Sample 3 was collected from this waste; therefore Sample 3 contained soluble proteins found in //E. Coli// that did not have an affinity for the Ni-NTA resin/buffer, or was lacking in histidine chains. Afterwards, a Wash (containing a scant amount of imidazole) was run through the column. This removed any weakly binding proteins. An Elution buffer, which contains a lot more imidazole, was then run through the column. The difference between Wash and Elution is the amount of imidazole in each. The Wash contains much less imidazole than Elution, and therefore only removes proteins that are weakly bound to Ni-NTA. The addition of Elution, in theory, removed a majority of pGEM-gbr22 from Ni-NTA. The runoff was collected, from which a small sample was taken. This was Sample 5, which should have contained a majority of the pGEM-gbr22 protein. Sample 6 was collected from a second run through of Elution buffer. Sample 6, contained the pGEM-gbr22 proteins that did not detach through the application of the first Elution buffer. Since imidazole has a stronger binding affinity to Ni-NTA than pGEM-gbr22, using Elution essentially removes the pGEM-gbr22.

The contents of Elution 1 were then analyzed using a Nanodrop spectrophotometer. Two readings were taken at 280 nanometers. Two more readings were taken at pGEM-gbr22’s optimal absorbance, 574 nanometers. The average for concentration for pGEM-gbr22 was 0.266525 mg/ml at 280 nanometers and 0.15808 mg/ml at 574 nanometers. 5.5 ml of pGEM-gbr22 was collected therefore the yield at 280 nm was 1.466 mg and the yield at 574 nm was 0.86944 mg. Less yield was found at the optimal wavelength, this may be attributed to contamination resulting in more protein being seen at 280 nm. The spectrophotometer may have detected other proteins other than pGEM-gbr22 at the optimal wavelength, but only if the other proteins have the same optimal wavelength. The difference in yield between 280 nm and 574 nm exposed the possibility of contamination.

The characterization of the proteins was done using SDS-PAGE electrophoresis. Samples 5 and 6 (in wells 6 and 7) should contain very few purple bands. The only band that should have been visibly present is the band of greatest intensity (pGEM-gbr22). While the band for Sample 5 is more intense than Sample 6, which is expected, there appears to be a lot of contamination in the two samples. The intensity of the protein in Sample 5 was estimated to be about 50% and Sample 6 was estimated to be about 75%. Each of the purple bands (aside from the most intense) represents proteins that were able to bypass the process of purification along with pGEM-gbr22. This could perhaps be attributed to histidine structures found in these proteins as well causing them to bind to the Ni-NTA beads. Another explanation may be that the imidazole used to make the Wash and Elution buffers were improperly made, since they were made at the beginning of the year. Comparing the most intense band against the ladder with the molecular weight standard, the molecular weight of pGEM-gbr22 was determined to be about 26 kDa. In actuality the molecular weight was 25.7942 kDa.

Sources of error for this experiment as mentioned earlier include perhaps faulty imidazole. Other errors such as accuracy of measurement may have played a result in the production of false data. Another error during the experiment occurred during the distributing of the samples into wells. The spectrophotometer may have also been used incorrectly resulting in data that seemed obscure. The cassette split between well one and two causing some of Sample 1 to leak over into the ladder during electrophoresis. Also despite staining for one and a half hours, the image is not as clear as it should be. This resulted in an image that was not as vivid as desired.

__ Conclusions: __

The experiment consisted of three parts. First the protein was expressed using //E. Coli// BL21. Then the protein was purified using Ni-NTA and Wash and Elution buffers, and the yield was determined. Finally the protein was characterized with an SDS electrophoresis. Despite the purification process, there was still contamination in the sample, however this may be attributed to error in the methods. Expression and purification may be used in Virtual Drug Screening to isolate a specific protein. Various ligands can then be tested against the protein to determine the inhibitive properties of various ligands against the desired proteins.

__ References: __


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 * 2) Weill Cornell Medical College. Studying Proteins and Protein Purification. http://www-users.med.cornell.edu/~jawagne/proteins_&_purification.html (accessed Apr 15, 2012).