RRC ID |
39324
|
著者 |
Yoneda A, Asada M, Suzuki M, Imamura T.
|
タイトル |
Introduction of an N-glycosylation cassette into proteins at random sites: expression of neoglycosylated FGF.
|
ジャーナル |
Biotechniques
|
Abstract |
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.
|
巻・号 |
27(3)
|
ページ |
576-8, 580, 582 passim
|
公開日 |
1999-9-1
|
DOI |
10.2144/99273rr05
|
PMID |
10489617
|
MeSH |
Amino Acid Sequence
Animals
Base Sequence
Blotting, Southern
Cell Line
DNA, Complementary / chemistry
DNA, Complementary / genetics
Deoxyribonuclease I / metabolism
Electrophoresis, Polyacrylamide Gel
Fibroblast Growth Factors / chemistry*
Fibroblast Growth Factors / genetics*
Gene Expression*
Glycosylation
Humans
Mice
Molecular Sequence Data
Mutagenesis, Insertional*
Sequence Analysis, DNA
Transfection
|
IF |
1.541
|
引用数 |
8
|
WOS 分野
|
BIOCHEMICAL RESEARCH METHODS
BIOCHEMISTRY & MOLECULAR BIOLOGY
|
リソース情報 |
ヒト・動物細胞 |
Ba/F3(RCB0805)
WEHI-3(RCB0035) |