Home › Immunology › Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation
Immunology JoVE (Open Access) Citable · DOI

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

DOI: 10.3791/60103-v
What you'll learn
  • ✓ Express and lyse E. coli cultures containing self-assembling protein nanoparticles
  • ✓ Purify and refold SAPNs using chromatography and denaturation-renaturation methods
  • ✓ Characterize nanoparticle size and homogeneity via DLS and gel electrophoresis
Protocol

A detailed method is provided here describing the purification, refolding, and characterization of self-assembling protein nanoparticles (SAPNs) for use in vaccine development.

Difficulty
advanced
Total time
~3–4 days (expression overnight, purification 1–2 days, refolding and characterization 1 day)
Biosafety
BSL-1

Steps

1
Lyse E. coli BL21(DE3) expressing SAPNs

Harvest induced E. coli cultures expressing six-helix bundle self-assembling protein nanoparticles and perform cell lysis to release target protein. Collect lysate for downstream purification.

▶ 00:43
2
Purify protein nanoparticles via chromatography

Apply lysate to chromatography columns to isolate and concentrate self-assembling protein nanoparticles from bacterial proteins and contaminants. Collect purified fractions.

▶ 01:24
3
Assess purity and identify protein composition

Analyze purified samples using SDS-PAGE gel electrophoresis and mass spectrometry to confirm protein identity and assess purity of nanoparticle preparations.

▶ 05:14
4
Refold denatured SAPN into native structure

Subject purified protein nanoparticles to controlled denaturation and refolding conditions to restore proper trimeric epitope presentation and quaternary assembly.

▶ 06:28
5
Measure nanoparticle size via dynamic light scattering

Perform DLS analysis on refolded SAPN samples to determine hydrodynamic diameter and verify successful self-assembly into uniform nanoparticles.

▶ 07:46
6
Interpret SAPN characterization results

Review representative DLS, gel electrophoresis, and mass spectrometry data to confirm successful production and correct nanoparticle size distribution for vaccine applications.

▶ 08:30
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