| RRC ID |
39324
|
| Author |
Yoneda A, Asada M, Suzuki M, Imamura T.
|
| Title |
Introduction of an N-glycosylation cassette into proteins at random sites: expression of neoglycosylated FGF.
|
| Journal |
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.
|
| Volume |
27(3)
|
| Pages |
576-8, 580, 582 passim
|
| Published |
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
|
| Times Cited |
8
|
|
WOS Category
|
BIOCHEMICAL RESEARCH METHODS
BIOCHEMISTRY & MOLECULAR BIOLOGY
|
| Resource |
| Human and Animal Cells |
Ba/F3(RCB0805)
WEHI-3(RCB0035) |
