Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Self-assembling protein nanoparticles, or SAPNs, can be used to develop vaccines against various infectious diseases. This method that we describe here can be used to purify, refold, and characterize SAPNs containing a six-helix bundle. This nanoparticle can present trimeric antigens in a native-like conformation.

With slight modifications, this method can be applied to other innovative SAPN vaccine constructs. Furthermore, it can be easily transferrable to large-scale production for clinical trials. For the lysis of harvested E.coli expressing the six-helix bundle SAPN, use a probe with the sonication output of 150 watts with four seconds of sonication and six seconds of rest, to sonicate resuspended bacterial pellets in 40 milliliters of imidazole-free buffer on ice for five minutes.

Centrifuge the cellular lysate to generate a clarified supernatant and transfer the supernatant to a sterile 150-milliliter flask. Then, bring the sample up to a final volume of 100 milliliters with fresh imidazole-free buffer. To isolate the protein of interest, open the FPLC software and click the New Method option.

In the Method Settings menu, open the Column Position dropdown menu and select C1 Port 3. In the Shown By Technique dropdown menu, select Affinity. In the Column Type dropdown menu, select Others, HisTrap HP, and and five milliliters.

The Column Volume and the Pressure boxes will automatically be set to the appropriate values. Click Method Outline and drag the Equilibration and Sample Application buttons, three Column Wash buttons, and the Elution button from the Phase Library popup menu next to the arrow before closing the Phase Library menu. Click Equilibration and set the values listed in the table to Initial Buffer B, 4%Final Buffer B, 4%and Column Volume, five.

Click Sample Application. In the Sample Loading box, click the radio button for Inject Sample on Column with Sample Pump, and confirm that the box next to the Use Flow Rate from Method Settings is checked in the Sample Injection with System Pump box. Change the Volume box value to 100 milliliters, and the Fraction Collection Scheme to Enable.

Unclick the Use Fraction Size from Method Settings box, and change the Fraction Size to four milliliters. Click on the first Column Wash button, set the values listed in the table to Initial Buffer B, 4%Final Buffer B, 4%and Column Volume, 10. Click the Fraction Collection Scheme Enable box and unclick the Use Fraction Size from Method Settings.

Change the Fraction Size to four milliliters, and click the second Column Wash button. Set the values in the table to Initial Buffer B, 0%Final Buffer B, 0%and Column Volume, five. Click the Fraction Collection Scheme Enable box and unclick the Use Fraction Size from Method Settings.

Change the Fraction Size to four milliliters, and click the third Column Wash button. Set the values in the table to Initial Buffer B, 0%Final Buffer B, 0%and Column Volume, 10. Click the Fraction Collection Scheme Enable button and unclick the Use Fraction Size from Method Settings box.

Then, change the Fraction Size to four milliliters. Click the Elution button and right click the information in the table. In the pop-up menu, click Delete Step and drag the Isocratic gradient button onto the table two times, so that there are two entries.

Set the value for the first entry to Initial Buffer B, 30%Final Buffer B, 30%and Column Volume, 10. Set the value for the second entry to Initial Buffer B, 100%Final Buffer B, 100%and Column Volume, 10. Click the Fraction Collection Scheme Enable button and click the Use Fraction Size from Method Settings box.

Then, click Save As and name the file purification. Place the pump A tubing of the FPLC into the imidazole-free wash buffer, and the pump B tubing into the 500-millimolar imidazole buffer. Place the sample pump tubing into the 100-milliliter sample and run the purification program.

When the 60%isopropanol wash is needed, pause the program, move the pump A tubing from the imidazole-free wash into the 60%isopropanol wash, and restart the program. At the end of the wash, pause the program again, return the pump A tubing to the imidazole-free wash buffer, and restart the purification program. To assess the purity of the purified protein, first combine the fractions from the flow through, wash one, wash two, and wash three steps, in individual 50-milliliter conical tubes.

Next, mix 15 microliters from each sample tube with 15 microliters of 2X Laemmli buffer in individual 0.5-milliliter microcentrifuge tubes and denature the samples at 95 degrees Celsius for 10 minutes. At the end of the incubation, set up a gel running apparatus with three stain-free four to 20%precast polyacrylamide gels in Tris-Glycine SDS-PAGE running buffer. Load eight microliters of molecular weight marker to the first well of each gel and 30 microliters of denatured sample to the other wells.

Run the gels at 200 volts until the dye front hits the bottom of the gel and briefly rinse the gels with deionized water before immediately imaging with a stain-free imaging system. Then, identify the fractions that contain protein bands with the correct size and pool these fractions. For six-helix bundle SAPN refolding, add 10 to 20 milliliters of pooled protein to a 10 kilodalton molecular weight cut-off dialysis cassette.

Dialyze the cassette in eight molar urea, 20-millimolar Tris, 5%glycerol, and five-millimolar TCEP at room temperature overnight. The next morning, decrease the urea concentration in the dialysis buffer stepwise by two molar every two hours to slowly dialyze the urea off the sample. When the urea concentration reaches two molar, move the dialysis apparatus to four degrees Celsius and dialyze the sample into 120-millimolar urea, 20-millimolar Tris, and 5%glycerol overnight to finish the refolding.

The next morning, remove the refolded protein from the cassette and filter the six-helix bundle SAPN through a 0.22-micrometer polyvinylidene fluoride syringe filter. To measure the mean particle size of the six-helix bundle SAPN, add 45 microliters of six-helix bundle SAPN to a disposable cuvette and place the cuvette in the DLS machine. Next, create a new measurement file and run a prewritten standard operating procedure for protein in 120-millimolar urea, 20-millimolar Tris, and 5%glycerol buffer at 25 degrees Celsius.

Then, start the measurement. The Z-average size, the PDI, and the distribution results will be calculated for each run. The machine will read the sample five times.

This fully assembled six-helix bundle SAPN is built upon protein sequences that are predicted to fold into a particle that contains 60 copies of the monomer. Total cell lysate was used to purify the six-helix bundle SAPN monomers by FPLC using a nickel column, demonstrating that the protein eluted at both 150 and 500-millimolar imidazole. The chromatogram also shows two other peaks at 185 and 210 milliliters total volume corresponding to the isopropanol wash and the imidazole-free wash respectively.

The fractions and the purity of the recombinant protein could be identified by gradient SDS-PAGE gels with the protein of interest primarily located in fraction 68 through 79. Western blot analysis with anti-His and anti-167D4 antibodies indicated that the pooled fractions were indeed the protein of interest. The blots also demonstrated the presence of the six-helix bundle SAPN multimers.

The samples that contained the protein monomers of interest were folded into the fully-assembled six-helix bundle SAPN by dialysis and particle size distribution was determined by DLS and nanoparticle tracking analysis. Visualization by transmission electron microscopy revealed well-formed individual six-helix bundle SAPN particles with the size distribution obtained from the two particle sizing techniques. Most important step in this protocol is the refolding of the six-helix bundle SAPN.

The correct pH and ionic strength of the refolding buffer are essential. Using an isopropanol wash during purification, we were able to reduce contaminating LPS to a very low level. However, LPS can be reduced further using an anion exchange column as necessary.

This technology can be easily adapted for human application. A bacterial-expressed SAPN has already been scaled up for an upcoming phase 1/2a malaria clinical trial.