This article describes a method for purifying recombinant proteins using vesicle formation promoted by the Vesicle Nucleating Peptide (VNp). The process involves culturing bacteria, inducing protein expression, and isolating vesicles containing the proteins for further analysis.
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, pellet the cells by centrifugation at 3000 g and 4 degrees Celsius for 20 minutes. Pass the supernatant through a sterile and detergent free 0.45 micron PES filter to sterilize the vesicle containing media for long-term storage.
To concentrate the vesicles into a smaller volume, pass the sterile vesicle containing media through a sterile and detergent free 0.1 micron MCE filter. Then, gently wash the membrane with 0.5 to 1 milliliters of sterile phosphate buffered saline, or PBS, and use a cell scraper, or plastic spreader to carefully remove vesicles from the membrane. Transfer the vesicle concentrate to a fresh microcentrifuge tube using a pipette.
Purified vesicles can be stored at 4 degrees Celsius. Sonicate the protein containing vesicles in the sterile media using an appropriate schedule to disrupt the vesicular lipid membranes and release protein. Centrifuge the sonicated suspension at 39,000 g and 4 degrees Celsius for 20 minutes to remove the vesicular debris.
繁體中文
