Cbl-b is a representative member of Ring E3 that regulates cytotoxicity of the natural killer (NK) cells. Cbl-b also ubiquitinates TAM family of RTKs including Tyro3, Axl and Mer on the surface of NK cells to unleash their killing instinct against cancer cells. I focused on using phage display to engineer the Ring domain of Cbl-b so it can function as an xE3 to exclusively transfer an engineered UB (xUB) through an engineered xE1-xE2 cascade to its substrate proteins. The development of an orthogonal UB transfer (OUT) cascade of Cbl-b will enable us to profile its direct ubiquitination targets. Additionally, I used in-vitro ubiquitination to get the inhibition of sulfono-γ-AA peptide, a new class of peptidomimetic foldamer, to mimic the α-1 helix of E2 and thereby inhibiting E1/E2 protein-protein interaction so as to disrupt the ubiquitination signaling cascade.