Purification of Self-Assembling Protein Nanoparticles using Affinity Chromatography

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)

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 (D....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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