|
KMID : 0364820100460010096
|
|
Korean Journal of Microbiology
2010 Volume.46 No. 1 p.96 ~ p.103
|
|
|
Comparison of Oligosaccharyltransferase Assay Methods Using a Fluorescent Peptide
|
|
Kim Seong-Hun
|
|
|
Abstract
|
|
|
Oligosaccharyltransferase (OTase) catalyzes the transfer of a lipid-linked oligosaccharide (LLO) to the nascent polypeptide. Most eukaryotes have an OTase composed of a multisubunit protein complex. However, the kinetoplastid Leishmania major and the bacterium Campylobacter jejuni have only a single subunit for OTase activity, Stt3p and PglB, respectively. In this study, a new in vitro assay for OTase was developed by using a fluorescent peptide containing N-glycosylation sequon, Asn-Xaa-Thr/Ser, where Xaa can be any amino acid residue except Pro. L. major Stt3p and C. jejuni PglB as a model OTase enzyme demonstrated the formation of glycopeptides from a fluorescent peptide through
OTase activities. For separation and measurement of the glycopeptides produced by the OTases, Tricine-SDS-PAGE, a lectin column and fluorospectrophotometer, and HPLC were applied. Comparison of these assay methods for analyzing a fluorescent glycopeptide showed HPLC analysis is the best method for separation of glycopeptides and nonglycosylated peptides as well as for quantify the peptides than other methods.
|
|
|
KEYWORD
|
|
|
fluorescent peptide, N-glycosylation, lipid-linked oligosaccharide, oligosaccharyltransferase, PglB, Stt3p
|
|
|
FullTexts / Linksout information
|
|
|
|
|
Listed journal information
|
|
|
|