ヒトノロウイルス突出ドメインの生産で

The overall goal of the protocol is the production and purification of human norovirus protruding domain in E.coli in order to obtain extremely pure protein for X-ray crystallography. Our protocol for protein expression, purification, and crystallization is fast, robust, and can be used for structural and nonstructural proteins. The advantage of this protocol is that our proteins are very pure and can form crystals that can diffract to high resolution.

According to our experience, the protocol might need adjustment for less soluble proteins. For example, the growth medium or growth temperature during protein expression can be changed. To begin, transform one microliter of the pMalc2x vector, coding for the MBP-His-P domain fusion protein into 15 microliters of competent E.coli BL21 cells using a standard transformation protocol.

Add 600 microliters of SOC medium to the cells. And grow for one hour at 37 degrees Celsius. Subculture the transformed cells into 120 milliliters of LB-ampicillin overnight at 160 rpm and 37 degrees Celsius.

Inoculate nine liters of LB-ampicillin with the subculture. Grow the cells in the incubator at 37 degrees Celsius with a shaking speed of 160 rpm until the optical density at 600 nanometers reaches 0.4 to 0.6. Subsequently, lower the temperature to 22 degrees Celsius for approximately one hour.

It is important to wait until the temperature reaches 22 degrees Celsius before inducing with a low amount of IPTG to allow for soluble protein expression. Induce the protein expression with 0.66 millimolar of IPTG. Following induction, grow the cells overnight at 22 degrees Celsius.

Harvest the cells by centrifugation. Discard the supernatant and freeze the cell pellet at 20 degrees Celsius. Prepare buffers that are used for the protein purification steps from stock solutions as detailed in the text protocol.

Thaw the cell pellet from the nine liter culture and dissolve in 150 milliliters of phosphate buffered saline at four degrees Celsius. Sonicate the cell suspension three times for two minutes to disrupt the cells. Centrifuge the sonicated cell suspension to separate cell debris from the supernatant containing expressed MBP-His-P domain fusion protein.

Collect the supernatant and discard the pellet. Wash and equilibrate a 10-milliliter slurry of nickel-NTA agarose beads with 10 millimolar imidazole buffer in a chromatography column. Add the equilibrated nickel beads to the supernatant containing expressed MBP-His-P domain fusion protein and incubate for 30 minutes at four degrees Celsius with slow rotation.

After incubation, apply the entire nickel bead-protein mixture to a chromatography column. Wash the column slowly with five CVs each of 10 millimolar, 20 millimolar, and 50 millimolar imidazole buffers at four degrees Celsius. Elute the MBP-His-P domain fusion protein using 250 millimolar imidazole buffer at four degrees Celsius.

During elution, check the optical density at 280 nanometers to verify the elution of the fusion protein. Continue the elution until the optical density at 280 nanometers drops to approximately 0.1. Working at four degrees Celsius, wash the beads with excessive amount of 250 millimolar imidazole buffer, followed by 10 millimolar imidazole buffer.

Save the beads for the second purification step. Verify the presence of the MBP-His-P domain fusion protein with sodium dodecyl sulfate polyacrylamide gel electrophoresis, or SDS-PAGE, using a 12%SDS polyacrylamide gel. Then concentrate the eluted MBP-His-P domain fusion protein to a final concentration of approximately three milligrams per milliliter.

The amount of protease needed to effectively cleave the MBP-His-P domain needs to be carefully calculated. It depends on the total amount of eluted fusion protein and on SDS-PAGE result. Cleave the MBP-His-P domain fusion with HRV 3C protease during dialysis against two liters of 10 millimolar imidazole buffer overnight at four degrees Celsius.

Depending on the final volume of concentrated protein, perform dialysis in a dialysis cassette or dialysis tubing. Equilibrate the nickel beads that were set aside in 10 millilmolar imidazole buffer. Incubate the dialyzed protein containing the cleaved P domain, MBP protein, and HRV protease with the equilibrated nickel beads for 30 minutes at four degrees Celsius with slow rotation.

Apply the nickel bead mixture to a column and collect the flowthrough at four degrees Celsius. This is the cleaved P domain. Measure the concentration of protein as it comes off the column until the optical density at 280 nanometers reaches approximately 0.1.

The MBP-His should remain bound to the nickel beads and the cleaved P domain is eluted in the flowthrough. Check the presence of cleaved P domain using SDS-PAGE with a 12%gel as before. After concentrating the eluted P domain to approximately three milligrams per milliliter, dialyze overnight at four degrees Celsius against gel filtration buffer for subsequent size exclusion chromatography purification.

Wash the pumps and pipes of the high performance liquid chromatography purification system, and pre-equilibrate the size exclusion chromatography column with gel filtration buffer. Inject the P domain onto the column at a flow rate of one milliliter per minute, using a superloop or loop, depending on the volume of the concentrated sample. After the injection is finished, increase the flow rate to 2.5 milliliters per minute.

As the optical density increases, and the P domain comes off the column, collect fractions of 1.5 milliliters. The P domain is usually eluted as a dimer. After checking the fractions using SDS-PAGE with a 12%gel, pool only the purest fractions and concentrate to approximately three milligrams per milliliter and approximately eight milligrams per milliliter.

Use the P domain at approximately three milligrams per milliliter and eight milligrams per milliliter for initial crystallization screening. Prepare at least 100 microliters of P domain per concentration for initial screens with 384 commercially available screening conditions. Perform screening at 18 degrees Celsius in a 96-well plate format where the reservoir contains 100 microliters of mother solution and a drop is composed of 0.2 microliters of mother solution and 0.2 microliters of protein.

Optimize the successful crystallization conditions using 15-well plates that contain three rows. Use 500 microliters of mother solution as a reservoir volume. Set up the first row with 100%mother solution.

The second row with 90%mother solution and 10%water. And the third row with 80%mother solution and 20%water. Set up the hanging drop with a drop size of two microliters by mixing an equal volume of protein and mother solution.

Use the optimized crystal conditions for co-crystallizing the P domain with ligands. Prepare plates as before but instead of a two microliter drop size, set up drops containing one microliter of mother solution, one microliter of protein, and one microliter of ligand at a concentration of one milligram per milliliter. Shown here is a representative SDS-PAGE result, showing the amount of eluted MBP-His-P domain fusion protein after the first purification step, where the MBP-His-P domain fusion protein is purified using nickel-NTA agarose beads.

Following separation of the MBP-His from the cleaved P domain, the amount of eluted cleaved P domain is also analyzed with SDS-PAGE. After the final purification step with size exclusion chromatography, crystal screening resulted in optimized crystal conditions. Here, the diffraction pattern of a protruding domain is shown.

In principle, it is possible to clone, expressed, purify, and crystallize proteins in less than four weeks, which makes this protocol a rapid system for analyzing newly emerging norovirus strains. While attempting this procedure, it is important to remember that quality is more important than quantity. Owing to the high quality of the purified P domains, additional studies are suitable, including immunization for antibody production, NMR experiments, and ELISA-based studies.

Moreover, the purified P domain can be used for complex formation with antibody fragments and nanobodies. After watching this protocol, you should have a good understanding of how to express and purify norovirus P domains of high quality.