Introduction of an N-glycosylation cassette into proteins at random sites: expression of neoglycosylated FGF.
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.
Yoneda
A
A
National Institute of Bioscience and Human Technology, Ibaraki, Japan.
Asada
M
M
Suzuki
M
M
Imamura
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D003850:Deoxyribonuclease I / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005346:Fibroblast Growth Factors / Q000737:chemistry* / Q000235:genetics*; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D017422:Sequence Analysis, DNA; D014162:Transfection
D018076:DNA, Complementary; D005346:Fibroblast Growth Factors; D003850:Deoxyribonuclease I