This method utilize the various way to tackle the recombinant viral protein expression challenges using one viral protein P'as a chaperone for another viral protein N'to obtain RNA-free N proteins. The RNA-free RSV N protein is obtained before assembling the virus specific RNA into nucleocapsid in vitro. The method can also be used with other non-segmented negative sense RNA viruses.
The ligation independent cloning method is a quick technique for acquiring co-expression constructs. Why or electives stain electron microscope is a quick method for checking large assemble in raw cell. We want to give you two tips.
First, get high yield and solid results for each step before moving to the next step. Second, set up proper programs for chromatography and practice picking up grids with forceps. For bi-cistronic construction of the N and P co-expression, grow four one liter cultures of E coli BL21DE3 strain cells in LB medium at 37 degrees Celsius.
When the optical density at 600 nanometers reaches 0.6, lower the temperature to 16 degrees Celsius. After one hour, induce protein expression by treatment with 0.5 millimolar IPTG overnight. The next morning, collect the cells by centrifugation and resuspend the pellets in 200 milliliters of lysis buffer.
lyse the cells by sonication for 15 minutes, with three seconds on, three seconds off pulses. And collect the lysates by centrifugation. Load the supernatant into a 2.5 by 10 centimeter cobalt gravity column, with approximately 10 milliliters of beads.
Pre-equilibrated with 5 to 10 column volumes of lysis buffer. And wash the column with five column volumes of buffer B and five volumes of buffer C.Use two volumes of buffer D to elute the protein from the beads. And equilibrate the Q column with five one milliliter volumes of QA buffer.
Use a peristaltic pump to load the diluted sample onto the column. Melt the loaded Q column onto the HPLC machine, along with fresh QA and QB buffers. Run the pump wash to successfully wash the machine with one to two volumes of QB and QA buffers.
After the last wash, set the flow rate to one milliliter per minute, and set UV1 to 280 nanometers and UV2 to 260 nanometers, using a 96 deep well plate to collect the fractions. Then use a stepwise gradient application of three to four volumes of each concentration of elution agent to elute the proteins, starting at a 0%QB concentration and increasing the percentage by 5%each time. The N-0 P protein complex will elute at the 15%QB buffer concentration.
Isolate the protein by gel filtration, in a one by 30 centimeters small scale purification column, equilibrated with buffer E.Then analyze the protein containing fractions by SDS page. For virus specific nucleocapsid assembly, mix and incubate the purified N-0 P complex with RNA oligo at a 1:1.5 molecular ratio at room temperature for one hour. Pre-equilibrate a small scale purification gel filtration column with buffer E.And remove any precipitation by centrifugation.
Load the supernatant, to the size exclusion chromatography column. And compare the size exclusion chromatography images of the protein to RNA assembly, and protein alone control samples, combining the nucleic acid purity ratios, to identify which peaks are the assembled N RNA, N-0 P and free RNA. To assemble an N RNA complex, collect all of the N RNA peak fractions, perform an RNA extraction, and run a urea page gel to double-check the length of the specific RNA.
To prepare a negative stain grid for negative stain electron microscopy, Use a heat plate to heat 5 mL of double distilled water to boiling and add 37.5 milligrams of uranyl formate to the water to obtain a 0.75%uranyl formate staining solution. Transfer the solution to an aluminum foil covered beaker. And add four microliters of 10 molar sodium hydroxide.
After 15 minutes of stirring protected from light, filter the solution through a 0.22 micron filter into a test tube. To make the continuous carbon coated electron microscopy grids hydrophilic, place the grids in a chamber connected to a power supply and apply negatively charged ions to the grids. Cut and fold a two by two inch parafilm strip.
Add two 40 microliter drops of distilled water to one end of the strip. And two 40 microliter drops of 0.75%uranyl formate staining solution to the other end. Add three microliters of protein sample to each grid.
After one minute, block the grids against blotting paper and dip the grids two times in the distilled water droplets. And once in the first staining solution droplet. Then immerse the grid in the second staining solution droplet for 30 seconds, before blotting the grids against blotting paper to remove any excess stain solution and allowing the grids to air-dry before imaging.
Using this protocol, a large-scale soluble hetero dimeric respiratory syncytial virus N0 P complex can be obtained. In E coli, the full length of N and N-terminal portions of the P proteins are co-expressed with a 10 X His tag on the N protein. Based on the UV absorbance nucleic acid purity ratio, N0 P contained both the full length N and N-terminal P, but did not contain cellular RNA.
The purified N0 P could then be stimulated and assembled into nucleocapsid like particles on electron microscopy grids via incubation with specific RNA oligos as demonstrated. When purifying the protein avoid air bubbles during the column purification and dilute the sample with QA buffer to adjust the pH and salt concentration before loading the sample onto the column. Take care to hold the electron microscopy grids at the outer edge to avoid contaminating the grids.
This will help us to determine the non-positive RSV genome packing questions, whether it is left handed or right handed helical structure. Following those procedures and authentic an RNA template for the RSV polymerase activity assay can be performed. And this RSV viral specific nucleocapsid can be used for the high resolution cryo EM analysis.
