Fig. 4: Staged expression of Rs Rubisco and QtEnc is essential for producing functional CO2-fixing compartments.
From: Reprogramming encapsulins into modular carbon-fixing nanocompartments
a Overview of producing Rs Rubisco (L8S8 control) and QtEnc-Rs Rubisco compartments in E. coli using co-induced (Co-I) or staged (ST) QtEnc expression strategies. Coloured circles indicate the genes being induced at each step. b Coomassie-stained blue native PAGE gel for analysis of E. coli total soluble protein and (c) corresponding Rs Rubisco immunoblot (following in-gel SDS denaturation prior to nitrocellulose transfer) enable comparison of RsL8S8 and RsL8Sc8 production and the formation of QtEnc-Rs Rubisco complexes following Co-I and ST QtEnc expression. Each experiment was repeated independently at least twice with similar results. d SDS-PAGE, 14CABP binding and kcatc of the purified QtEnc, Co-I and ST complexes, with the Rs Rubisco produced in tobRsLS leaves32 provided as a positive control for quantitative measurements and subunit stoichiometry. Ratio of measurements between samples is shown as bubble plots, with relative RbcL and RbcS content between samples determined by gel densitometry on Coomassie-stained bands, along with quantification of purified QtEnc concentration against a BSA standard. e Schematic representation drawn to scale, depicting the contents of purified QtEnc Co-I and ST compartments. The Co-I sample contains unassembled RsRbcSc and inactive RsL?Sc? subcomplexes of unknown oligomeric state, while the CO2-fixing ST sample contains functional RsL8Sc8, as well as a minor amount of unassembled RsRbcSc. Source data are provided as a Source Data file.
