Recombinant Protein Production and Recovery from Bacteria-Derived Vesicles

Abstract

Source: Streather, B. R., et al. Optimized Production and Analysis of Recombinant Protein-Filled Vesicles from E. coli. J. Vis. Exp. (2023)

This video demonstrates the production and isolation of recombinant proteins tagged with Vesicle Nucleating Peptide (VNp) from bacterial vesicles. An inducer triggers VNp-tagged protein expression, driving vesicle formation and encapsulation, followed by centrifugation and sonication for efficient protein recovery without cell lysis

Protocol

1. Bacterial cell culture and protein induction

1. To induce recombinant protein expression from the T7 promoter, add isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of up to 20 µg/mL (84 µM). The induction of recombinant protein expression must occur at the late-log phase (i.e., typical optical density, OD₆₀₀ of 0.8-1.0) for the production of vesicles. ​
   NOTE: The length of the induction period may differ between proteins, with some reaching maximum production at 4 h and others overnight (18 h). To date, maximum vesicle export has been obtained in overnight cultures.

2....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Isopropyl β-D-1-thiogalactopyranoside (IPTG)	Melford	367-93-1
Lysogeny Broth (LB) / LB agar	Lab Stock	NA	10 g/L Tryptone; 10 g/L NaCl; 5 g/L Yeast Extract (1.5 g/L agar)
MF-Millipore Membrane filter (0.1 µm, MCE)	Merck	VCWP04700
Millipore Express PLUS membrane filter (0.45 µm, PES)	Merck	HPWP04700
Phosphate buffered saline (PBS)	Lab Stock	NA