Effects of N-butyldeoxynojirimycin and the Lec220.127.116.11 mutant phenotype on N-glycan processing in Chinese hamster ovary cells: application to glycoprotein crystallization
Butters TD, Sparks LM, Harlos K, Ikemizu S, Stuart DI, Jones EY, Davis SJ. (1999), Protein Sci. 8, 1696-701
Heterologous gene expression in either (1) the glycosylation-defective, mutant Chinese hamster ovary cell line, Lec18.104.22.168, or (2) the presence of the α-glucosidase inhibitor, N-butyldeoxynojirimycin facilitates the trimming of N-linked glycans of glycoproteins to single N-acetylglucosamine (GlcNAc) residues with endoglycosidase H (endo H). Both approaches are somewhat inefficient, however, with as little as 12% of the total protein being rendered fully endo H-sensitive under these conditions. It is shown here that the combined effects of these approaches on the restriction of oligosaccharide processing are essentially additive, thereby allowing the production of glycoproteins that are essentially completely endo H-sensitive. The preparation of a soluble chimeric form of CD58, the ligand of the human T-cell surface recognition molecule CD2, illustrates the usefulness of the combined approach when expression levels are low or the deglycosylated protein is unstable at low pH. The endo H-treated chimera produced crystals of space group P3121 or P3221, and unit cell dimensions a = b = 116.4 Å, c =51.4 Å α = β = 90°, γ = 120°, that diffract to a maximum resolution of 1.8 Å.