Figure 2

Purification of N-glycan untrimmed and trimmed tag-free hFasLECDs. a) Elution profile of N-glycan untrimmed tag-free hFasLECD sample (pre-purified by 1st Hi-Trap S cation-exchange chromatography) in 2nd cation-exchange chromatography. Used column: Resource S 6 ml. The region shown in underbar was collected and used for characterization in c). NaCl concentration under principal peak eluting condition is described. b) Elution profile of N-glycan trimmed tag-free hFasLECD sample in 3rd cation-exchange chromatography. Used column: Mono S 1 ml. The region shown in underbar was collected and used for characterization in c). NaCl concentration under principal peak eluting condition is described. c) Elution profiles of fractionated products in size-exclusion chromatography. Solid line: N-glycan untrimmed tag-free hFasLECD. Dashed line: N-glycan trimmed tag-free hFasLECD. Used column: Superdex 200 10/300 GL. Elution buffer: 50 mM sodium acetate plus 150 mM NaCl (pH 5.6). Flow rate: 0.5 ml/min. The peak retention time of each sample is described. Vertical arrows indicate the elution positions of molecular-weight size-markers [Ald, aldolase (158 kDa); Ova, ovalbumin (44 kDa); Rna, ribonuclease A (13.7 kDa) under the same conditions. d) SDS-PAGE analysis of purification course during N-glycan trimming with Endo Hf glycosidase and receptor-mediated co-immunoprecipitation using wild type and mutant hFasRECD-T-Fcs. Lanes: a, N-glycan untrimmed tag-free hFasLECD; b, after Endo Hf digestion; c, after Con A column fractionation; d, after Mono S column fractionation; e, co-immunoprecipitated materials using wild-type hFasRECD-T-Fc [15]; f, co-immunoprecipitated materials using hFasRECD-T-Fc (N102Q, N120Q) mutant; M, Molecular-weight markers. “+CHO” and “ΔCHO” indicate the migration positions of N-glycan untrimmed and N-glycan trimmed tag-free hFasLECD, respectively.