Introduction of an N-Glycosylation Cassette into Proteins at Random Sites: Expression of Neoglycosylated FGF
| Autor: | Atsuko Yoneda, Masahiro Asada, Masashi Suzuki, Toru Imamura |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 1999 |
| Předmět: | |
| Zdroj: | BioTechniques, Vol 27, Iss 3, Pp 576-590 (1999) |
| Druh dokumentu: | article |
| ISSN: | 1940-9818 0736-6205 |
| DOI: | 10.2144/99273rr05 |
| Popis: | 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. |
| Databáze: | Directory of Open Access Journals |
| Externí odkaz: |
|