| Products/Services Used | Details | Operation |
|---|---|---|
| Gene Synthesis | The gene encoding for enhanced green fluorescent protein (EGFP) with hexapeptide KEGYES fused to C-terminus was synthesized and subcloned into vector pET28a-His by GenScript (Nanjing, China). The DNA sequence of Engineering human Ubch5c and S. cerevisiae Ubc4 were synthesized by GenScript and further cloned into the vector pET28a-His. The DNA sequence of human p53, S. cerevisiae Ufd2 and Ufd4 were synthesized and inserted between the BamHI and EcoRI sites of the vector pGEX-4T-1 with an N-terminal GST tag followed by an HRV 3C protease cleavage site by GenScript. The genes encoding for human α-synuclein,human cyclin B1 (1-88, also known as NCB1) with His tag fused to Cterminus, and human NEDD8 were synthesized and subcloned into vector pET28a by GenScript. | Get A Quote |
Ubiquitination, catalyzed usually by a three-enzyme cascade (E1, E2, E3), regulates various eukaryotic cellular processes. E3 ligases are the most critical components of this catalytic cascade, determining both substrate specificity and polyubiquitination linkage specificity. Here, we reveal the mechanism of a naturally occurring E3-independent ubiquitination reaction of a unique human E2 enzyme UBE2E1 by solving the structure of UBE2E1 in complex with substrate SETDB1-derived peptide. Guided by this peptide sequence-dependent ubiquitination mechanism, we developed an E3-free enzymatic strategy SUE1 (sequence-dependent ubiquitination using UBE2E1) to efficiently generate ubiquitinated proteins with customized u... More
