We are trying to understand the dynamic interactions between molecules within living cells. For biochemical analysis, it can be necessary to use large quantities of recombinant protein. These can be extremely difficult to express in a correctly folded and functional form.
If a protein was found to be misfolded or insoluble when expressed in bacteria, researchers would either have to resort to mammalian cells, which is expensive and relatively slow, or use synthetic polypeptides. Our protocol allows for high yield expression, as well as export of challenging proteins from E.coli. Adding a short cleavable peptide sequence to the amino terminus of the protein not only enhances the production and yield of every protein we have tested to date, but also exports the protein from the cell into the media.
Along with simplifying protein expression, it also simplifies downstream protein purification. After cloning the vesicle nucleating peptide, or VNP, sequence tag at the amino terminal of the fusion protein culture a 5 milliliter lysogeny broth, or LB, starters from fresh bacterial transformations at 37 degrees Celsius to saturation. Then use that to inoculate 25 milliliters of terrific broth, or TB, containing appropriate antibiotic selection in a 500 milliliter conical flask, Incubate the flask in an incubator at 37 degrees Celsius while shaking at 200 rotations per minute, or greater than equal to a 25 millimeter orbital throw until the culture reaches a 600 nanometer optical density value or OD 600 of 0.8 to 1.0.
Then, 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 x 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 x g and 4 degrees Celsius for 20 minutes to remove the vesicular debris. BL21 DE3 Escherichia coli containing the VNP 6 mNeonGreen expression construct displayed mNeonGreen fluorescence being induced overnight with IPTG.
The fluorescence remained visible in the culture after the removal of bacterial cells by centrifugation. The presence of VNP mNeonGreen within the culture in cleared culture media was confirmed by sodium dodecyl sulfate, polyacrylamide gel electrophoresis. The resuspended purified vesicles in PBS also displayed fluorescence.
To stain the vesicle membrane add the fluorescent lipid dye FM4-64 to the purified vesicles at a final concentration of 2 micromolar and incubate for 10 minutes before imaging. After isolating the protein filled vesicles from Escherichia coli culture pipette the purified vesicles onto a less than 1 millimeter thin circular 2%LB agarose pad that has been allowed to form and set on a clean glass slide. Once the liquid has dried place a 50 millimeter x 25 millimeter cover slip over the vesicles on the pad.
Hold the cover slip in place with spacers and adhesive tape. Then mount the slide onto an inverted microscope using an oil immersion objective, and leave the sample for 2 to 3 minutes to settle and the temperature to equilibrate. For single frame images, use three image averaging to reduce hardware dependent random background noise.
For time-lapse imaging allow 3 to 5 minutes between individual frames. Widefield fluorescence microscopy imaging showed the purified amnion green containing vesicles. The lipophilic fluorescent dye FM4-64 confirmed the presence of vesicle membranes.
Structured illumination microscopy of the live bacterial cells expressing the inner membrane protein CydB fused to mNeonGreen and VNp6-mCherry2 showed vesicle production and cargo insertion.
