Expression and Purification of Recombinant Vibrio cholerae σ38, σ54 and RNAP: Insights into Homotypic Oligomerization and Holoenzyme Assembly Barriers.

Alternative sigma factors such as σ38 (RpoS) and σ54 (RpoN) are essential for the environmental adaptation and pathogenesis of Vibrio cholerae, yet remain poorly characterized. In this study, we report a recombinant expression and purification pipeline for the V. cholerae alternative sigma factors and RNA polymerase (RNAP) core using affinity chromatography, ammonium sulfate precipitation and size-exclusion chromatography (SEC). SEC and Native PAGE analysis reveal that σ38 & σ54 exist as stable, homotypic oligomers (tetramer and trimer, respectively), while the RNAP core forms dimeric and higher-order assemblies. The oligomeric assemblies may cause a significant biophysical barrier to in vitro holoenzyme reconstitution due to the burial of core-binding surfaces within the oligomer interface. Our results suggest a model of convergent evolution where structurally unrelated sigma factors are sequestered in stable storage modes, providing a critical technical baseline for understanding the specialized activation requirements of the Vibrio cholerae transcriptional machinery.