Recombinant Protein Production and Recovery from Bacteria-Derived Vesicles

Samenvatting

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. Recombinant vesicle isolation

1. Pellet the cells by centrifugation at 3,000 x g (4 °C) for 20 min.
2. To sterilize vesicle-containing media for long-term storage, pass the cleared culture media through a sterile and detergent-free 0.45 µm polyethersulfone (PES) filter.
   NOTE: To test the exclusion of viable cells from the vesicle-containing filtrate, plate onto lysogeny broth (LB) agar and incubate overnight at 37 °C.
3. To concentrate vesicles into a smaller volume, pass the sterile vesicle-containing media through a sterile and detergent-free 0.1 µm mixed cellulose esters (MCE) filter.
4. Gently wash the membrane with 0.5-1 mL of sterile phosphate-buffered saline (PBS) using a cell scraper or plastic spreader to carefully remove vesicles from the membrane. Transfer to a fresh microfuge tube. ​
   NOTE: Purified vesicles can be stored in sterile media or PBS at 4 °C. There are examples of recombinant proteins stored in these vesicles for 6 months, in this way, with no loss in enzymatic activity.

3. Soluble protein release from isolated vesicles

1. Once protein-containing vesicles have been isolated into the sterile media/buffer, subject vesicular lipid membranes to sonication using an appropriate schedule for the apparatus (e.g., 6x 20 s on and off cycles) and centrifuge at 39,000 x g (4 °C) for 20 min to remove vesicle debris. NOTE: Osmotic shock or detergent treatment can be used as an alternative to break open the vesicles, but consideration must be given to the impact upon protein functionality and/or downstream application.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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