Expression of human perlecan domain I as a recombinant heparan sulfate proteoglycan with 20-kDa glycosaminoglycan chains |
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Authors: | Graham L D Whitelock J M Underwood P A |
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Institution: | CSIRO Molecular Science, Sydney Laboratory, North Ryde, New South Wales, 1670, Australia. lloyd.graham@molsci.csior.au |
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Abstract: | Recombinant forms of human perlecan domain I were secreted as proteoglycans by stably transfected human 293 cells. A recombinant domain I-only proteoglycan spanned the 95- to 265-kDa region in SDS-PAGE and appeared to be 160 kDa in denaturing gel filtration. Its glycosaminoglycan (GAG) content was approximately 67% heparan sulfate, and its average GAG chain size of 20 kDa suggested that the true molecular mass of the proteoglycan was 90 kDa. Domain I with enhanced green fluorescent protein fused to its C-terminus had an apparent molecular mass of 210-220 kDa and contained approximately 100% heparan sulfate. Its average GAG chain size (also 20 kDa) suggested a true molecular mass of 117 kDa for this proteoglycan. Its sulfate content of 53-77 mol SO2-4 per mole of protein indicated the presence of one sulfate group per 4-7 GAG sugar residues. |
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