| Products/Services Used | Details | Operation |
|---|---|---|
| Gene Synthesis | The plasmid encoding S. cerevisiae Ufd4 gene was synthesized by GenScript (Nanjing, China), and its codon was optimized for S. cerevisiae and Escherichia coli (E. coli) overexpression, respectively. For overexpression by S. cerevisiae, the Ufd4 gene was subcloned to the vector YEplac181 containing an N-terminal DYKDDDDK (Flag) tag. For overexpression by E. coli, the gene was inserted between the BamHI and EcoRI sites of the vector pGEX-4T-1 with an N-terminal GST tag followed by an HRV 3 C protease cleavage site. The plasmid encoding human TRIP12 (442–1992) was also synthesized by GenScript, and its codon was optimized for HEK293F overexpression, which was subcloned to the vector pcDNA3.1 containing an N-terminal Flag tag. The DNA sequence encoding yeast (S. cerevisiae) Ubc4 was synthesized by GenScript and its codon was optimized for E. coli overexpression. The gene was further cloned into the vector pET-28a with an N-terminal His tag followed by an HRV 3 C protease cleavage site. Different Ufd4 and Ubc4 mutants were constructed by site-directed mutagenesis. The DNA sequences of wild-type Ub and Ub mutants including Ub-K6R, UbK11R, Ub-K27R, Ub-K29R, Ub-K33R, Ub-K48R, Ub-K63R, Ub-G76C, UbK29R-G76C, Ub-K29R/K48R, Ub-K29C/K48R, Ub-77D (a Ub variant with an additional amino acid Asp at the C terminus), Ub-K29C/77D and UbMCQ (Ub variant with an additional amino acid Cys between Met1 and Gln2) were constructed and cloned to the vector pET-22b by GenScript. The plasmids containing S. cerevisiae/human Uba1, S. cerevisiae Ubc2/human Ubch7, S. cerevisiae Ubr1, and the truncated S. cerevisiae genes Scc1 (268–384; also known as MCD1) were the same as previously used. | Get A Quote |
| Codon Optimization | Get A Quote | |
| PCR Cloning and Subcloning | Get A Quote | |
| Mutagenesis Services | Get A Quote |
The K29/K48-linked ubiquitination generated by the cooperative catalysis of E3 ligase Ufd4 and Ubr1 is an enhanced protein degradation signal, in which Ufd4 is responsible for introducing K29-linked ubiquitination to K48-linked ubiquitin chains to augment polyubiquitination. How HECT-E3 ligase Ufd4 mediates the ubiquitination event remains unclear. Here, we biochemically determine that Ufd4 preferentially catalyses K29-linked ubiquitination on K48-linked ubiquitin chains to generate K29/K48-branched ubiquitin chains and capture structural snapshots of Ub transfer cascades for Ufd4-mediated ubiquitination. The N-terminal ARM region and HECT domain C-lobe of Ufd4 are identified and characterized as key structural... More
