Expression and purification of recombinant Vibrio cholerae σ³⁸, σ⁵⁴ and RNAP: Insights into homotypic oligomerization...

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.