Source: Karch, C. P. et al., Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation. J. Vis. Exp. (2019)
This video showcases the purification of histidine-tagged self-assembled protein nanoparticles or SAPNs using affinity liquid chromatography. The resulting purified SAPN fractions are suitable for vaccine development, holding promise for potential immunotherapeutic applications.
1. Expression of the Self-assembling protein nanoparticles core containing a six-helix bundle (SHB-SAPN) Protein in E. coli BL21(DE3)
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.
3. Protein purification using a His-column
NOTE: This protocol was performed using an FPLC instrument, but it can be adapted to gravity flow.
The authors have nothing to disclose.
2-propanol | Fisher | BP26181 | 4 L |
Ampicillin | Fisher | BP1760-25 | 25 g |
Corning Disposable Vacuum Filter/Storage Systems | Fisher | 09-761-108 | A variety of sizes |
GE Healthcare 5 mL HisTrap HP Prepacked Columns | GE HealthCare | 45-000-325 | 5 pack |
Glycerol | Fisher | BP229-4 | 4 L |
Imidazole | Fisher | O3196-500 | 500 g |
Magic Media E. coli Expression Medium | ThermoFisher | K6803 | 1 L |
MilliporeSigma Millex Sterile Syringe 0.22 mm Filters | Millipore | SLGV033RB | 250 pack |
One Shot BL21 Star (DE3) Chemically Competent E. coli | ThermoFisher | C601003 | 20 vials |
Sodium Phosphate Monobasic | Fisher | BP329-500 | 500 g |
Tris Base | Fisher | BP152-1 | 1 kg |
Urea | Fisher | BP169-500 | 2.5 kg |
Equipment | |||
Fiberlite F14-14 x 50cy Fixed-Angle Rotor | ThermoFisher | 096-145075 | Rotor |
NGC Quest 10 Chromatography System | BioRad | 7880001 | FPLC to aid in protein purification |
Sonifer 450 | Branson | also known as 096-145075 | Sonicator |
.