Title: The Purification and Characterization of Over-expressed Bacterial Protein BL21(DE3)

Introduction:

In recent years the use of recombinant proteins in scientific research has increased greatly, as has the wealth of techniques and products used for their amplification and purification. [1] The techniques of protein expression, purification, and characterization are especially important to the field of virtual drug screening in that they are heavily involved in the wet lab portion of the research. The expression of the protein usually involves genetically modified plasmids that are inserted into human, bacterial, plant or animal cells in which they are overexpressed. Purification of these proteins often involves enzymes such as lysozymes which can digest cell walls and extract the protein. The characterization part of these labs show the purity of the protein and the overall success of the expression. Characterizing the purified protein in some detail reduces the risk of wasting resources on protein material of inadequate quality. It also provides a means to ensure that different batches of the same protein have similar properties [2]. This lab aims to use the techniques of protein expression, purification, and characterization to obtain a relatively concentrated and pure elution of pGEM-gbr22, a protein found in coral with a distinctive purple color. If methods are followed properly the protein elution should be purple and its characterization should indicate a heavy, pure concentration of protein.

Materials & Methods:

During expression of BL21(DE3), two samples were iced with plasmid pGEM-gbr22, heat shocked at 42 degrees Celsius for 45 seconds, and returned to ice. 200 ul of SOC was added to the solutions, which were then shaken in an incubator for 30 minutes, and spread on separate LB/amp agar plates with colirollers. The next morning, one bacterial colony was transferred to LB/amp media to shake in the incubator for 8 hours. The starter culture was then moved into an LB/amp flask for further growth. Later, the sample was spun to isolate the pellet, and then resuspended in PBS buffer. For purification, lysozyme and cyanase were added to the bacteria, the soluble fraction was filtered through a 0.22 syringe filter PES with a 5 mL syringe, and purified using a combination of batch and column chromatography. NiNTA resin buffer was used as the 1st rinse through the bio-rad and collected as “Waste”. A wash buffer containing 0.2 mL imidazole was used for the 2nd rinse and collected as “Wash.” An elution buffer containing 2.5 mL imidazole was used for the 3rd and 4th rinse and collected as “Elution 1” and “Elution 2.” The Nanodrop spectrophotometer was used at 280 nm and 574 nm (the maximum absorbance wavelength), along with the extinction coefficient and Beer’s law, to find the concentration and yield of the protein in “Elution 1”. Samples for each step in the process of purifying were collected and labeled for a total of 6 samples. In characterization, the samples taken throughout expression and purification were heated and centrifuged with loading buffer. Sample 1 was centrifuged for 5 minutes at 5,000 rpm to retain the pellet, which was suspended in 200 ul of water and added to 40 ul of loading buffer. 10 ul of loading buffer was added to Samples 2 to 6. All samples were placed into a heat block at 95°C for 5 minutes and centrifuged for 2 minutes at 5,000 rpm. The samples were then loaded into individual gel cells, and subjected to an electric current in an electrophoresis tank. The gel was then cleaned in nanopure water and stained with imperial protein stain on an orbital shaker. After destaining, the gel was dried on the drying bed, documented, and analyzed.

Results:

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Figure 1: Ampicilin positive agar control plate with no BL21(DE3) bacterial growth

exhibited in the morning on Day 2 after 24 hour incubation at 37 degrees Celsius.

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Figure 2: Ampicilin positive agar experimental plate with colonies of BL21(DE3)

bacteria transformed with plasmid DNA pGEM-gbr22 exhibited ) in the morning

on Day 2 after 24 hour incubation at 37 degrees Celsius.

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Figure 3: Ampicilin negative agar “fun plate” (coughed on) in the morning on

Day 2 after an incubation period of 24 hours at 37 degrees Celsius.

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Figure 4: A large culture of BL21(DE3) bacterial cells, transformed

with plasmid DNA pGEM-gbr22, in the evening on Day 3 after

spending 24 hours in the shaking incubator at 37˚C and 250 rpm.

(does this have amp?)

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Figure 5: A wet pellet of 0.32g of BL21(DE3) bacterial cells

transformed with plasmid DNA pGEM-gbr22 in the evening

on Day 3 after centrifuging for 10 minutes at 4°C and 5,000 rpm.

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Figure 6: 5 micro-liters of Elution 1 of the protein after purification.

Gbr22 protein after washing with 1x PBS and 250mM imidazole solution.

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Figure 7: 5 micro-liters of Elution 2 of the protein after purification.

Gbr22 protein after washing with 1x PBS and 250mM imidazole solution.

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Figure 8: Absorption vs. Wavelength at 280 nm wavelength of elution 1 sample. Blue line represents the absorbance reading.

The Beer’s Law formula A=Ebc was used to find the concentration. The absorbance value, the extinction coefficient and the cuvette path length were put into the formula. This equation looks like 0.332 = (38850)(1)(c). The concentration wass found to be 8.546e-6 Mol/L. Multiplying this concentration by the molar weight of the protein (25794.2 g/Mol), the yield was found as 0.2204 mg/mL. Since there is 5 mL, the final yield was 1.102 mg.

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Figure 9: Page Ruler Prestained Protein Ladder Product 26616 Thermoscientific used in this lab for gel electrophoresis.

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Figure 10: Gel after drying on Whitman filter paper, covered with Saran wrap. The lane farthest to the left is the protein ladder. Lanes 2-7 consist of samples 1 through 6. Sample 5, the elution wash, shows about 80% purity. The protein is estimated to be about 25 kDa.

Discussion:

There are two enzymes used in the lab that serve to extract the protein from the bacteria. The lysozyme is used to digest the cell wall of the bacteria, and cyanase is used to digest the DNA and RNA in the mixture. The six histidine residues attach at the C-terminuses (end of the protein) to separate the protein from other things in the solution. Nickel metal binds to the Histine residues not allowing the protein to go through the column until the elution buffer is used. Sample one was cell lysate so protein was present. Sample two was SOL (what is this acronym?)and a small amount of protein was present. Sample three was a flow through; no protein (the protein being worked with) should have been present. Sample four was wash buffer and no protein should have been present just the other substances in the solution. Sample five was elution one and should only have the protein and sample six should have whatever protein was left in the column. The buffer is supposed to remove everything else but the protein being worked with. While the elution buffer has imidazole that competes with the histidines to elute the protein off the beads. The size of the protein determined for the gel is 25 kDa which was very similar to the size determined in the protein purification lab. The purity of the protein is 80% because there was only one clear, dark band, and a one very faint one. The protein stayed in the filter until the elution buffer was used. The 6xHis tag and the protein stuck to the nickel. The protein sticks to the Ni-NTA beads and the nickel (Ni-NTA implies the presence of nickel)so it doesn’t flow through the filter until elution buffer.

Possible sources of error for this lab could include inaccurate measurements of chemicals used, as well as not waiting enough time, or waiting too much time, between steps.

Conclusions:

The three labs of protein expression, purification, and characterization lead (led*)to the gel that, when analyzed, showed that there was 80% purity. With this data it could be assumed that the labs were successful. Initially, the target protein was inserted in the bacteria, overexpressed, and then purified using elution 1 and 2. The Nanodrop and Beer’s Law were then used to find the concentration. In the protein characterization lab the gel showed that Sample 5 was for the most part one dark purple line, hence the high purity percentage. The molecular weight based on the ladder was found to be 25 kDa, which is similar to the concentration found from the purification lab, showing the labs did work. All the steps learned from this lab are very important later on when more target proteins will need to be harvested, characterized, and used for further testing in independent projects. Using virtual drug screening programs like GOLD combined with PyMol, ligands can be found that bind to the protein to make a drug inhibitor and then tested in the wet lab. (not necessarily for this lab, it is just a model protein whose functionality we don't necessarily care to inhibit)

References:

[1] Graslund, S.; Nordlund, P., Weigelt, J., et. al, Protein production and purification. Nat Methods 2008, 5 (2), 135-46

[2] Mattaj, I. W. Protein Expression and Purification Core Facility. http://www.embl.de/pepcore/pepcore_services/index.html (accessed April 16).