Fig. 1. (A) Schematic representation of the α1AGP? in this study.The 90 amino acid sequences were deleted from C-terminus ofhuman α1AGP. Artificial N-glycosylation sites (2N) were addedN-terminal of 93 amino acid sequences of deleted α1AGP. (B) 151Amino acid sequences of the recombinant α1AGP?. 30K signalpeptides and 2N sites were added in N-terminus, and Histidine-tag (His x 8) and TEV protease cleavage site in C-terminus. Theasparagine residues (N) to which N-glycan may be added indicatedby bold letters. The amino acid numbers of asparagine as N-glycansites is shown at the bottom.
Fig. 2. Expression of the recombinant α1AGP? in cultured silkwormcells. Time courses of the expression of the α1AGP protein inBme21 cells, BmN4 cells and BmN4 SID-1 cells (A). The cells andculture medium were collected at 2, 3, 4 days post-infection (DPI).The recombinant α1AGP? was detected by Western blotting usingHis-Probe.
Fig. 3. (A) Purifcation of human α1AGP from larval haemolymph.The Histidine tagged α1AGP? protein was purifed through nickelaffinity chromatography as described in Materials and methods.Each fraction was resolved on 15% SDS-PAGE and visualized byCoomassie Brilliant Blue (CBB) R-250. IP: input; FT: fow-throughfraction; W: wash fraction; Lane No.1~3: eluent fraction (100mMimidazole); Lane No.4~9: eluent fraction (500mM imidazole). (B)Characterization of N-glycan structures of the α1AGP secreted insilkworm larval haemolymph. The purified recombinant α1AGPor α1AGP? form silkworm larvae as indicated in Materials andmethods were incubated with (+) or without (?) PNGaseF for 1 hat 37 °C. After reaction, each mixture was resolved on 15% SDS-PAGE and visualized by CBB R-250, His-Probe, or Concanavalin A(ConA).
Fig. 4. (A) The purifed α1AGP? was separated into multiple bandsin 15% SDS-PAGE and visualized by CBB R-250. The multiplebands were assigned Band 1 ~ Band 8 from the top. Band 1 is thesmear portion at the top of Band 2. (B) The degree of glycosylationof each band 1~ 8 analyzed by LC/MS. At the schematic diagram ofN-glycan, the open square, open circle and flled triangle representGlcNAc, mannose and fucose, respectively. The frequency ofaddition of N-glycans at each site is indicated by shading of thediagrams. Approximate number of sugar chains attached to eachband is shown on the right.
Table 1. The ratio of N-glycan structure for each sugar chain binding site.
Table 1. Continued
Table 1. Continued
Table 1. Continued
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