LscB, LscBUpNA and LscBUpA showed levan formation. (b) Schematic representation of the DNA fusion products. The dashed line and dashed arrow represents lscB while the solid line and solid arrow represents lscA. Characterization of lsc fusion proteins To verify the molecular sizes of Lsc encoded by the individual fusion constructs, a Western blot analysis
using Lsc-specific antibodies was performed (Figure 3a). Under denaturing conditions, it was interesting to observe that LscBUpNA migrated at an intermediate rate i.e. faster than LscB but slower than LscBUpA. The signal for LscBUpA was weaker than those representing LscB or LscBUpNA suggesting that the N-terminus of LscB might contribute to the expression level or stability of Lsc. In contrast, protein samples of PG4180.M6 transformed with LscA or LscAUpB did not show any signal specific for Lsc at all
thus confirming that lack of levan formation was due to lack of the corresponding protein. Venetoclax in vitro Figure 3 Detection of levansucrase. (a) Western blot analysis: 10 μg of total proteins were separated by 10% SDS-PAGE, transferred onto PVDF membrane, hybridized with anti-Lsc antiserum and detected using BCIP/NBT. The dark bands (arrow) correspond to Lsc and the corresponding fusion proteins. (b) Zymogram: 100 μg of total proteins were separated by 10% native-PAGE and incubated in 5% sucrose solution overnight. The white bands indicate formation of levan after utilization of sucrose by Lsc and the fusion proteins. To check for the enzymatic Dabrafenib purchase function of Lscs encoded by the individual fusion constructs, zymographic detection was done with non-denatured total protein samples of transformed mutants (Figure 3b). The above reported levan forming ability of transformants M6(lscB), M6(lscBUpNA) and M6(lscBUpA) could be attributed
to the enzymatic functioning of proteins or fusion proteins. As expected, native protein samples derived from M6(lscA) or M6(lscAUpB) did not exhibit any in-gel levan Abiraterone production (Figure 3b). An interesting observation was the altered electrophoretic mobility of the enzymatically active proteins. The LscBUpNA migrated slower as compared to LscB even though the predicted molecular masses of both proteins were almost identical (~47.6 kDa) suggesting possible differences in the respective protein charges. In accordance with the Western blot results, LscBUpA seemed to be less expressed than LscB or LscBUpNA suggesting an important role of the N-terminus for transcriptional or translational processes. MALDI-TOF analysis The altered electrophoretic migration rate of LscBUpNA as compared to LscB during the native gel protein separation suggested that the two proteins were indeed different although their predicted protein sizes were almost identical. To demonstrate that LscBUpNA produced a unique and novel enzyme and to show that the other two transformants indeed also produced the intended Lsc proteins, we subjected the levan-forming fusion proteins to MALDI-TOF analysis.