TR reporters were designed pairwise for either secretion or cell membrane integration through the inclusion of the C-terminal SEA and transmembrane domain of MUC1 and assorted with a comprehensive set of TR reporters. These transmembrane TR reporters were expressed transiently in glycoengineered HEK293 cells that do not appear to express endogenous Figure 1. Design of the human mucin tandem repeat (TR) cell-based display platformIllustration of the mucin TR display approach with membrane-bound and secreted mucin reporters expressed in KO/KI glycoengineered isogenic HEK293 cell lines. HEK293 wild-type (WT) cells are predicted to produce a mixture of mSTa, dST, and sialylated core2 structures, and through stable genetic engineering, a library of isogenic HEK293 cells with different O-glycosylation capacities was developed. The library enables display of mucin TRs with different O-glycan structures as indicated (glycan symbols and genetic design shown), as well as tunable site occupancy by the engineering of the GALNT isoenzyme gene repertoire (left part). The secreted mucin reporter design contains an N-terminal 6xHis and FLAG tag, and GFP followed by different mucinTR domains of ca. 200 amino acids (single TR domains used for MUC3, MUC5B, MUC13, MUC6, and GP1bα), and a second C-terminal 6xHis tag. The membrane-bound mucin reporters contain an N-terminal FLAG tag and GFP followed by the mucin TR domain and further includes the SEA and transmembrane domain of human MUC1 in the C-terminal end for membrane retention. The most characteristic TR sequence for each reporter is illustrated with the number of TRs included (right part). Transient or stable expression of the mucin TR reporters in the glycoengineered isogenic HEK293 cell library enables the display of cell surface mucin TRs as well as production of secreted mucin TRs with distinct O-glycan structures. Structures of glycans are shown with symbols drawn according to the Symbol Nomenclature for Glycans (SNFG) format. Adopted from Nason et al,.2021, Nature Commun.natural context of glycoproteins and the cell surface. Utilizing this glycoengineered cell library platform, we generated a cell-based array for cell surface display of representative regions of human mucin TRs. Figure 1 presents an overview of the concept for the cell-based display and production of human mucin TR reporters with programmed O-glycan structures. The mucin 113
元のページ ../index.html#113