Human 90K (h90K; Mac-2-binding protein) glycoprotein is a potential pharmaceutical due to its inhibitory activity against cancer metastasis and expansion. Here, h90K glycoprotein was produced in insect Drosophila S2 cell system, and its N-glycan pattern was analyzed. A plasmid encoding h90K gene, fused with a hexahistidine tag under the control of Drosophila metallotionein promoter, was stably transfected into S2 cells. After copper sulfate induction, transfected S2 cells secreted recombinant h90K at a good expression level of 28 mg/L in a 150-mL spinner flask culture. The purified recombinant h90K showed an apparent molecular weight of similar to 78 kDa which was much smaller than that (similar to 97 kDa) of the natural h90K. Because de-N-glycosylated h90K appeared at similar to 60 kDa protein band, it was suggested that the recombinant h90K from S2 cells has small N-glycans with about half the molecular weight (similar to 18 kDa) of N-glycans of the natural h90K. Through detail analyses using high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, the S2-derived recombinant h9OK was confirmed that it has simple paucimannosidic structures containing two or three mannose residues with core fucose as the major (similar to 79%) N-glycans. (C) 2013 Elsevier Inc. All rights reserved.