The overall goal of this procedure is to express surface proteins on mycobacterium using surface decoration with recombinant proteins as an alternative to traditional, genetic-based approaches. These methods can help answer important research questions in the field of mycobacteriology, especially in the area of hospital interactions and the vaccine developments. This technique can be used to screen for potential interference factors or antigen candidates.
It can be the and rapidly compared to fastidious and time-consuming traditional genetic-based approaches. Demonstrating this procedure will be Amina Talal, a research assistant in my lab. This portion of the protocol begins with transforming P17 Aviden OVA plasmid into E.Coli BL21.
To do so, induce 250 milliliters of E.Coli BL21 culture with IPTG for three hours at 37 degrees Celsius. After three hours, lyse the bacteria to solubilize the inclusion bodies by first pelleting the induced BL21 culture and discarding the supernatant. Then re-suspend the pellet in 10 milliliters of lysis buffer and allow the cells to be lysed for 15 minutes.
Then continue the incubation at room temperature using a rotator for agitation, and let it go overnight. In the morning, centrifuge the mixture for half an hour. Then collect the supernatant containing the solubilized inclusion bodies.
Next purify the Aviden OVA proteins using a nickel NTA column. First, dilute the supernatant one to two in lysis buffer and estimate the volume. Then prep%re a column containing four milliliters of nickel NTA resin per 250 milliliters of diluted supernatant and load the dilution onto the column.
Next, wash the column three times using 10 milliliters of lysis buffer at pH seven per wash. To collect the recombinant protein, wash the column with lysis buffer containing imidazole and collect the eluate. If your recombinant protein is free from inclusion bodies, the following is procedure that will good for many proteins in our lab.
First, adjust the pH of the refolding solution to be one unit above or below the protein's isoelectric point. Then, gradually vortex and dilute the protein to a 1 to 10 dilution in refolding solution. Then incubate the protein on a magnetic stirrer for 30 minutes at room temperature to complete the refolding process.
Next, de-salt and buffer exchange the refolded proteins into PBS using a protein concentrator. Follow by centrifuging the column and store aloquats of soluble protein at 20 degrees Celsius. Work in a biosafety cabinet using personal protective equipment because BCG is a level 2 pathogen.
To biotinylate the surface of BCG cells, first grow the cells on a shaker platform to the proper density. Collect about one billion cells and wash them three times using 500 microliters of ice-cold, endotoxin-free PBST. Then, spin down the cells, and suspend them in sterile, endotoxin-free PBS.
Next, prepare fresh 10 millimolar sulfo-NHSSS biotin in sterile filtered water. Then incubate bacteria in one milliliter of broth containing 0.5 millimolar of sulfo-NHSSS Biotin. Perform the incubation at room temperature for 30 minutes.
Next wash the now labeled bacteria three times with 500 microliters of ice-cold PBST, as before to remove the un-reacted re-agent. Then, re-suspend the pellet in one milliliter of PBST. To evaluate the biotinylation efficiency, stain an aloquat of 100 million biotinylated BCG with 25 microliters of Strepravidin-FITC.
Then, briefly incubate the stained bacteria in para-formaldehyde, and then measure the level of biotinylation using flow cytometry. After assessing the growth of and survival of the biotinylated mycobacteria, proceed with binding the Aviden fusion protein to them. First, mix 500 million biotinylated BCGs with Aviden fusion protein for a final concentration of 10 micrograms of protein per milliliter.
Let this reaction go for an hour at room temperature on a shaker. Next, wash the bacteria three times with 500 microliters of ice-cold PBST. Then stain the bacteria with rabbit anti-Aviden antibody for 20 minutes at room temperature.
After 20 minutes, perform one wash step with 500 microliters of ice-cold PBST. Then, stain the bacteria with FITC-conjugated goat anti-rabbit IGG antibody. Next, wash the bacteria using 500 microliters of ice-cold PBST, and analyze the bacteria by flow cytometry to evaluate the surface decoration.
After following the described protocol, cytology shows that there were minimal levels of OVA binding to non-biotinylated bacteria and significant levels of OVA binding to biotinylated bacteria. Importantly, the levels of Aviden OVA on the bacterial surface remained stable and detectable one month after lyophilization and storage at room temperature. Next, RAW cells and dsRed bacteria were used to perform a phagocytosis assay.
Pleasingly, there was no deterrence in the macrophage phenotype. To exam Aviden OVA decorated BCGs fate within macrophages, adherent RAW cells were infected and examined by florescence microscopy. Aviden OVA was not just present on the bacterial surface, but also detached and trans-located to cytoplasm far from the bacteria.
An immuno-gold staining experiment showed that Aviden OVA antigens can detach from the BCG surface, cross the phagosomal membrane and travel toward the cytosol. Macrophages infected with Aviden OVA BCG showed co-localization of Avi OVA with IA molecules. This suggests that Aviden fusion proteins detach and trans-locate to antigen presentation compartments to be loaded onto MHC2 molecules.
Aviden OVA peptides also co-localized with class I molecules within BCG phagosomes, as shown by H2K staining. Thus, there is potential presentation of Avi OVA antigen to CD8 positive T-cells by the macrophage. After watching this video, you should have a good understanding of how to express proteins in mycobacteria, and this organism hard to manipulate with current genetic methods.
After the technique, we use it successfully to load the vaccine with the mycobacteria antigen is at six. Then we showed that the modified vaccines was opportunity to use as specific immuno response in the mouse model. We are currently collaborating with to explore this approach in teratine We proposed to use BCG upgraded with selected antigen or immunobulators.
