Extended Data Fig. 2: Expression, structure, and dimerization of the catalytic domain of human FUT9.
From: Structural basis for Lewis antigen synthesis by the α1,3-fucosyltransferase FUT9
(a) Diagrammatic representation of the protein coding regions from the FUT9-pGEn2 and FUT9-pGEn3 expression constructs. The fusion protein from the FUT9-pGEn2 construct contains an NH2-terminal signal sequence followed by an 8xHis tag, AviTag, superfolder GFP, TEV protease cleavage site, and the catalytic domain of FUT9 containing three N-glycan consensus sequons sites at N62, N101, and N153. The protein coding region for the FUT9-pGEn3 construct is identical to the FUT9-pGEN2 construct with the exception of the inclusion of an Ig Fc domain between the GFP domain and TEV protease cleavage site. (b) Expression of the recombinant product in HEK293S (GnTI-) cells resulted in secretion of the fusion protein into the culture medium (Crude media), and subsequent Ni2+-NTA purification yielded a highly-enriched enzyme preparation (IMAC1 elution). Cleavage of the enzyme with TEV protease and EndoF1 resulted in removal of the tag sequences and glycans, and further purification by Ni2+-NTA chromatography and Superdex-75 (Superdex-75 gel filtration) led to the final purified preparation. Each transfection experiment was performed at least three times and two different SDS PAGE gels of the samples were generated. Data presented are representative of the respective experiments Original uncropped images are provided in the Source Data. (c) The purified FUT9 catalytic domain was further characterized by size exclusion-multiangle light scattering (SEC-MALS). A280 is shown by the green line, refractive index in blue, light scattering in red and calculated molar mass in black. The molecular mass derived from SEC-MALS analysis (~77 kDa) is in close agreement with a dimeric form of the FUT9 catalytic domain monomer following cleavage with TEV and EndoF1 (~37.8 kDa).
