Results

Fluorescence assay of supernatants derived from strains with single copy genes

The first obstacle that we had to overcome was establishing a suitable protocol to boost the secretion capacities of our generated Bacillus subitilis producer strains. After initially testing various media and incubation periods we performed all experiments using 2xYT medium and an incubation time of 16 h.

Applying this protocol, we observed good fluorescence in the supernatants harvested from the eight strains (combinations of fluorescence proteins (FP) with SpyTag/SpyCather (Tag) constructs) secreting either mCherry or sfGFP. We were able to detect higher fluorescence when compared to supernatants derived from the wild-type (Figure 4).

The supernatant of the wild-type shows a relatively high fluorescence in case of sfGFP , because of the autofluorescence derived from the medium (data not shown). When compared with each other, all of the different fusion possibilities of a FP and the Tags show a similar fluorescence, with the exception of the N-terminally fused mini. SpyCatcher constructs. Apparently, this fusion either inhibits the native folding of the FP or decreases the secretion efficiency.

To check the efficiency of the secretion process, we also determined the fluorescents of the cells. The supernatant was separated from the cells by centrifugation.

The pelleted cells were resuspended in fresh medium, immediately followed by a fluorescence measurement (assuming the recorded fluorescence exclusively derives from FPs still inside the cells). We compared the relative fluorescence of both samples and also included untreated 16h old cell culture in this assay.

Figure 5 shows data generated for one strain (mCherry-mini. SpyCatcher) as an example. While the relative fluorescence in the unprocessed cell culture and the supernatant do not differ significantly, the emission of the fresh-resuspended cells show a clear drop of fluorescence (comparable to the wild-type level).

This clearly demonstrates that indeed the measured fluorescence intensities derive from secreted FPs.

Fluorescence assay of supernatants derived from strains with multi copy genes:

To further increase the secretion capacity, three constructs were sub-cloned into the pBS0E vector which is a medium copy number B. subtilis specific vector. When performing the same fluorescence assay as bevor, we obtained eight to ten times higher fluorescence intensities in the supernatants when compared to the supernatants derived from single copy (Figure 6).

The sfGFP fusion proteins were visible in the supernatant when excited with blue light (Figure 7) and the typical red colour of mCherry could even be observed under daylight conditions (Figure 8). These protein rich supernatants were used for stability tests, spectral analysis, purification and SDS-PAGE.

Stability:

After incubating the supernatants containing the fluorescent fusion protein for 24h they still showed the same fluorescence intensity, implying that the protein is quite stable in 2xYT medium (graph included in pdf below). Using the B. subtilis strain WB800N as host contributes to an increased stability, as it is not secreting any proteases that may degrade the protein of interest.

Emission spectra:

The emission spectra of the supernatants containing fusion protein producing cultures were analysed to prove that the measured fluorescence’s originates from sfGFP or mCherry. In Figure 9 the emission spectra of the sfGFP-fusion protein is clearly visible, having an emission peak at ca. 510 nm. The mCherry-fusion protein displays the correct emission spectrum as well, having a peak at 615 nm.

The 2xYT medium that was used for cultivation shows over all high auto-fluorescence when sfGFP wavelengths were measured. As already mentioned bevor, this influences the total fluorescent emissions of the supernatant. Likewise, it needs to be considered that the wildtype supernatant has a lower fluorescence than the medium, suggesting a quenching effect caused by B. subtilis. On the other side, only very low auto-fluorescents or quenching effects could be observed when the mCherry spectrum was recorded. Thus, we decided to carry on with mCherry constructs to prove the applicability of the SpyTag/SpyCatcher systems.

Purification and SDS-PAGE:

By running SDS-PAGE with the purified supernatants of mCherry fusion protein producing cultures, we could prove the functionality of the SpyTag/SpyCatcher partners. In Figure 10 it is clearly visible, that the supernatants from the production strains (lane 4 and 6) contain protein bands of high concentration that the wild-type (lane 2) lacks. These proteins band are showing up again in the elution after purification, proving that the bands are referring to the his-tagged target proteins, mCherry-mini. SpyCatcher having a weight of 36,6 kDa and mCherry-SpyTag weighting 31,9 kDa.

Surprisingly the supernatant with mCherry-SpyTag contained a band of the wrong weight, and the mCherry-mini. SpyCatcher even three bands the wrong weight, indicating accumulation or degradation of the protein of interest.

None the less upon combining the two supernatants and incubating for 4 h, a new protein band with ca. 70 kDA shows up (lane 8 and 9). This is the definite proof, that the mini. SpyCatcher and the Spytag are functional and mediated the covalent bonding of the mCherry constructs.

Conclusion

Our team succeeded in the production of self conjugating protein complexes using B. subtilis secretion capacity’s. Even though the system could not yet be tested in Peptidosomes, we are very much sure that our vision to facilitate the production process of tuneable protein complexes can be achieved. Peptidosomes as a novel platform for cultivation and B. subtilis powerful secretion are a promising combination that should be studied further.

References