Recombinant Protein Production and Recovery from Bacteria-Derived Vesicles

Begin with a culture of a bacterial species that encodes recombinant proteins tagged with the Vesicle Nucleating Peptide, or VNp, a short peptide that promotes vesicle formation.

Add an inducer to the culture and incubate. The inducer triggers protein expression.

VNp embeds in the inner membrane and promotes membrane curvature, encapsulating the VNp-tagged proteins into budding vesicles.

Transfer the vesicle-rich medium to a tube. Centrifuge it to separate the vesicle-rich supernatant.

Next, filter the supernatant to remove residual contaminants.

Then, pass the filtrate through another membrane filter to capture vesicles on the membrane surface.

Place the membrane in a dish and add phosphate-buffered saline.

Gently detach the vesicles and transfer the suspension into a clean tube.

Sonicate the suspension to disrupt vesicle membranes and release the proteins.

Centrifuge the lysate to separate membrane fragments.

The resulting supernatant contains purified recombinant proteins, ready for analysis.

Induce recombinant protein expression from the T7 promoter by adding IPTG to a final concentration of up to 20 micrograms per milliliter, or 84 micromolar. After allowing sufficient time for induction