Introduction of an N-glycosylation cassette into proteins at random sites: expression of neoglycosylated FGF
Yoneda, A.; Asada, M.; Suzuki, M.; Imamura, T.
Biotechniques 27(3): 576-8 580 582 Passim
1999
ISSN/ISBN: 0736-6205 PMID: 10489617 Document Number: 505108
We developed a method for introducing an N-glycosylation cassette into proteins at random sites by constructing cDNAs and expressing it in mammalian cells. The protocol entails four steps: (i) generation of cDNAs that contain single, randomly-located blunt end cuts; (ii) ligation of N-glycosylation cassettes into the blunt end cuts in three-frame formats; (iii) selection of the cDNA clones encoding N-glycosylated proteins; and (iv) subcloning into an expression vector for transfection and expression in mammalian cells. This method was evaluated using secreted fibroblast growth factor (FGF) as a model protein. Several secreted FGF cDNA clones, each containing an AsnLeuSer-coding sequence at a random site, were obtained. When these clones were expressed in mammalian cells, some of the secreted FGFs were found to be N-glycosylated. The method described here should also be applicable for random introduction of functional oligopeptide/polypeptide cassettes into virtually any protein of interest.