Purification of Self-Assembling Protein Nanoparticles using Affinity Chromatography

1. Expression of the Self-assembling protein nanoparticles core containing a six-helix bundle (SHB-SAPN) Protein in E. coli BL21(DE3)

1. Mix 95 mL of component A and 5 mL of component B of the media in a 2 L sterile glass Erlenmeyer flask as per the manufacturer's instructions (see the Table of Materials). Add ampicillin to a final concentration of 100 μg/mL.
2. Inoculate the media with E. coli from a previously established glycerol stock culture. Incubate culture at 30 °C with shaking at 200 rotations per minute (rpm) for 48 h.
   NOTE: The used E. coli BL21 (DE3) stock contained the ampicillin resistant expression vector with the SHB-SAPN gene. Although the general protocol of the media recommends 24 h of incubation at 37 °C, 48 h of incubation at 30 °C gives higher yield for SHB-SAPN.
3. Transfer the culture to two 50 mL conical tubes. Centrifuge the tubes at 4,000 x g for 10 min with a fixed angle rotor at 4 °C. Remove the supernatant and save the pellet to harvest cells.
   NOTE: The cell pellet can be either processed immediately or frozen at -80 °C until use.

2. Lysis of E. coli BL21(DE3) by sonication

NOTE: Use nonpyrogenic plasticware and glassware baked at 250 °C for at least 30 min. Tris(2-carboxyethyl)phosphine (TCEP), as a reducing agent, breaks the disulfide bonds within and between proteins. TCEP is necessary for the buffers during this protocol if the displayed antigen contains S-S bonds. For the SHB-SAPN core only, the presence of TCEP in the buffers is not essential.

1. Prepare imidazole-free buffer (8 M Urea, 50 mM sodium phosphate monobasic, 20 mM Tris base, 5 mM TCEP) pH 8.0 (adjusted with 5 N sodium hydroxide, NaOH) and filter it using a 0.22 µm vacuum bottle filtration unit.
2. Resuspend the pelleted cells (from step 1.3) with 40 mL of imidazole-free buffer in one 50 mL conical tube. Sonicate the resuspended cells with a probe on ice for 5 min (4 s of sonication, 6 s of rest) with a sonication output of 150 W.
3. Centrifuge the cellular lysate (40 mL) at 29,000 x g at 4 °C for 25 min in a fixed angle rotor to generate clarified supernatant. Transfer the supernatant to a 150 mL sterile flask and discard the pellet. Dilute the supernatant to 100 mL using the imidazole-free buffer (later in the protocol referred to as "sample").
   NOTE: This dilution step is needed to prevent the fast performance liquid chromatography (FPLC) system pressure from becoming too high during lysate loading on the column.

3. Protein purification using a His-column

NOTE: This protocol was performed using an FPLC instrument, but it can be adapted to gravity flow.

1. Prepare the following buffers and filter them using a 0.22 µm vacuum bottle filtration unit: (i) imidazole-free buffer "Buffer A" (8 M Urea, 50 mM sodium phosphate monobasic, 20 mM Tris base, 5 mM TCEP) pH 8.0; (ii) 500 mM imidazole buffer "Buffer B" (8 M Urea, 50 mM sodium phosphate monobasic, 20 mM Tris base, 5 mM TCEP, 500 mM Imidazole) pH 8.0; and (iii) isopropanol wash (20 mM Tris, 60% isopropanol) pH 8.0.
   NOTE: pH for each buffer was adjusted with 5 N NaOH.
2. Equilibrate the His-column.
   NOTE: For lab scale production, this protocol uses a 5 mL prepacked His-column, but any larger sized column can be used.
   1. Open the FPLC software and click on the New method option. It will immediately open to the Method settings menu. Under the drop-down menu for column position choose C1 port 3.
   2. On the Shown by technique drop down menu choose affinity. On the Column type drop-down menu choose others, Histrap HP, 5 mL. The column volume and the pressure boxes will be automatically set to the appropriate values.
   3. Click on the Method outline button. Drag the following buttons from the Phase library popup menu: equilibration, sample application, column wash, and elution next to the arrow in that exact order. Close the Phase library menu.
   4. Click on the equilibration button. The values listed in the table should be "initial buffer B" (4%), "final buffer B" (4%), and "volume (CV)" 5.
   5. Click on the Sample application box. In the sample loading box click the radio button for Inject sample on column with sample pump. Make sure the box next to the Use flow rate from method settings is checked in the sample injection with system pump box. Next to the volume box on the right side of the screen change the value to 20 mL.
   6. Click on the Column wash button. The values listed in the table should be "initial buffer B" (4%), "final buffer B" (4%), and "volume (CV)" 5. Next to the fraction collection scheme unclick the Enable box.
   7. Click on the elution button. The values listed in the table should be "initial buffer B" 0%, "final buffer B" 100%, and "volume (CV)" 5. Next to the fraction collection scheme, click on the Enable box. Unclick the Use fraction size from method settings and adjust the fraction size to 4 mL in the fill-in box below.
   8. Click the save as button on the top of the software. Name the file "equilibration."
   9. Connect a 5 mL prepacked His-column to the corresponding column port 3 on the FPLC. Both pump A and pump B tubing, as well as the sample pump tubing, should be placed into 0.22 µm filtered deionized water. Run the equilibration program.
   10. Place both pump A and pump B, as well as the sample pump tubing, into the imidazole-free buffer (Buffer A) and run the equilibration protocol again.
3. Bind the sample to the column and purify the protein.
   1. Open the FPLC software and click on the New method option. It will immediately open to the Method settings menu. Under the drop-down menu for Column, position choose C1 port 3. On the Shown by technique drop-down menu, choose affinity. On the column type drop-down menu choose others, Histrap HP, 5 mL. The column volume and the pressure boxes will automatically be set to the appropriate values.
   2. Click on the Method outline button. Drag the buttons from the Phase library popup menu: equilibration, sample application, column wash (Wash 1), column wash (Wash 2), column wash (Wash 3), and Elution next to the arrow in that exact order. Close the Phase library menu.
   3. Click on the Equilibration button. The values listed in the table should be "initial buffer B" 4%, "final buffer B" 4%, and "volume (CV)" 5.
   4. Click on the Sample application box. In the sample loading box, click the radio button for the Inject sample on the column with the sample pump. Make sure the box next to the Use flow rate from method settings is checked in the sample injection with the system pump box.
   5. Next to the volume box on the right side of the screen, change the value to 100 mL. Next to the fraction collection scheme, click the Enable button. Unclick the Use fraction size from the method settings box and then change the fraction size to 4 mL.
   6. Click on the first column wash button (Wash 1). The values listed in the table should be "initial buffer B" 4%, "final buffer B" 4%, and "volume (CV)" 10. Next to the fraction collection scheme, click the Enable box. Unclick the Use fraction size from method settings and then change the fraction size to 4 mL.
   7. Click on the second column wash button (Wash 2). The values listed in the table should be "initial buffer B" 0%, "final buffer B" 0%, and "volume (CV)" 5. Next to the fraction collection scheme, click the Enable box. Unclick the Use fraction size from method settings and then change the fraction size to 4 mL.
   8. Click on the third column wash button (Wash 3). The values listed in the table should be "initial buffer B" 0%, "final buffer B" 0%, and "volume (CV)" 5. Next to the fraction collection scheme, click Enable. Unclick the Use fraction size from the method settings box and then change the fraction size to 4 mL.
   9. Click on the Elution button. In the table right, click the information listed and, on the menu that comes up, click the Delete step. Drag the isocratic gradient button onto the table twice so that there are two entries.
   10. The value for the first entry should read "initial buffer B" 30%, "final buffer B" 30%, and "Volume (CV)" 10. The value for the second entry should read "initial buffer B" 100%, "final buffer B" 100%, and "volume (CV)" 10. Next to the fraction collection scheme, click the Enable button. Click the box next to Use fraction size from method settings.
   11. Click the Save button on the top of the software. Name the file "purification." Pump A tubing of the FPLC should be placed into the imidazole-free wash buffer, while pump B tubing should be placed into the 500 mM imidazole buffer. The sample pump tubing should be placed into the 100 mL sample.
   12. Run the "purification" program and wait for the time when the 60% isopropanol is needed (Wash 2). Pause the program; move pump A tubing from the imidazole-free wash into the 60% isopropanol wash. Restart the program.
   13. Once the isopropanol step is completed, pause the program again and move to pump A tubing back into the imidazole-free wash buffer. Restart the purification program (the rest of the run is automated).