DAI LINGYUNSINGAPORE-MIT ALLIANCE2014-10-172014-10-172014-07-09DAI LINGYUN (2014-07-09). SORTASE A TRANSPEPTIDASE ENABLES SYSTEMATIC DISSECTION OF CD1-ASSOCIATED LIPID ANTIGENS. ScholarBank@NUS Repository.https://scholarbank.nus.edu.sg/handle/10635/99068CD1 COMPLEXES ARE SURFACE-ANCHORED GLYCOPROTEINS THAT PLAY AN INDISPENSABLE ROLE IN (GLYCO)LIPID ANTIGEN PRESENTATION DURING PATHOGENIC CHALLENGE. A FEW CD1-PRESENTED LIPID ANTIGENS HAVE BEEN DISCOVERED; HOWEVER, CURRENTLY CD1 COMPLEXES ISOLATION METHODS FOR LIPID DISSECTION ARE UNDERMINED BY INHERENT LIMITATIONS, CAUSING POTENTIAL BIASES. WE ENVISIONED AND EXPERIMENTALLY DEMONSTRATED A NOVEL APPLICATION OF SORTASE A (SRTA)-MEDIATED TRANSPEPTIDATION STRATEGY ON SHAVING OFF CD1 COMPLEXES FROM THE LIMITING CELL MEMBRANE. THE ENGINEERED CD1 COMPLEXES WERE SHOWN TO TRAVERSE NORMALLY THROUGH DESTINED ENDOSOMAL COMPARTMENTS FOR LIPID PRESENTATION. MOREOVER, WE PROVIDED THE PROTEIN PURIFICATION SCHEME FOR RELEASED CD1 COMPLEXES, PERMITTING SYSTEMATIC ANALYSIS OF THE BOUND LIPIDS. TOGETHER, WE MADE A TECHNICAL ADVANCE THAT FOR THE FIRST TIME PERMITTING EFFICIENT RELEASE OF MEMBRANE-ASSOCIATED CD1 COMPLEXES, WITH THE MERITS OF MINIMAL PERTURBATIONS OF PROTEIN CONFORMATION AND INTRACELLULAR TRenCD1 complex, Sortase A, Lipid antigen, Cell engineering, Mass spectrometry, Systems biologyThesisNOT_IN_WOS