Purification of Self-Assembling Protein Nanoparticles using Affinity Chromatography
Published: May 31, 2024
Abstract
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.
Protocol
1. Expression of the Self-assembling protein nanoparticles core containing a six-helix bundle (SHB-SAPN) Protein in E. coli BL21(DE3) 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. Inoculate the media with E. coli from a previously established glycerol stock cultur…
Açıklamalar
The authors have nothing to disclose.
Materials
| 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 |
Etiketler
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Bu Makaleden Alıntı Yapın
Purification of Self-Assembling Protein Nanoparticles using Affinity Chromatography. J. Vis. Exp. (Pending Publication), e22267, doi: (2024).