Fig. 5. Generating defined homo- and heterotypic polyUb-POI conjugates.

a Combining Ubl-tools with enzymatic Ub assembly allows generation of K48-tetraUb, site-specifically charged onto a protein of interest, here sfGFP. b SDS-PAGE analysis of enzymatically accessed and distinctly modified K48-linked diUb-A and diUb-B. Full gels as well as LC-MS analysis can be found in Supplementary Fig. 9b, c. c Incubation of diUb-A and diUb-B in the presence of Srt2A leads to formation of K48-tetraUb, as depicted by SDS-PAGE analysis and whose integrity is corroborated by LC-MS. Single asterisk (*) denotes hydrolysis of Srt2A recognition motif and cleavage of H₆-tag. Full gels and densitometrically determined yields can be found in Supplementary Fig. 10b. d SDS-PAGE analysis showing Srt5M-catalyzed charging of K48-linked tetraUb onto sfGFP-N150GGK. Densitometric analysis revealed formation of tetraUb-sfGFP formation in 45% yield. e Schematic representation showing Ubl-tools generated branched Ub chains (K11/K48 branched) and their site-specific attachment to a POI. f SDS-PAGE analysis displaying the formation of K11/K48-branched triUb by incubation of Ub-K11/K48GGK with Ub(LAT) and Srt2A. Double asterisks (**) denote an impurity in the Srt2A-stock. Full gels and densitometrically determined yields can be found in Supplementary Fig. 13b. g Integrity of purified K11/K48-branched triUb is shown by SDS-PAGE analysis and LC-MS. h Incubation of K11/K48-branched triUb with sfGFP-N150GGK in presence of Srt5M leads to the formation of defined branched triUb-sfGFP conjugate. Single asterisk (*) denotes hydrolysis of Srt5M recognition motif and cleavage of H₆-tag. Densitometric analysis revealed branched triUb-sfGFP formation with 33% yield. Consistent results were obtained over at least three replicate experiments. Source data are provided as a Source Data file.