RRC ID |
30956
|
著者 |
Srichaisupakit A, Ohashi T, Fujiyama K.
|
タイトル |
Identification of a protein glycosylation operon from Campylobacter jejuni JCM 2013 and its heterologous expression in Escherichia coli.
|
ジャーナル |
J Biosci Bioeng
|
Abstract |
Campylobacter jejuni is a human enteropathogenic bacterium possessing an N-glycosylation system. In this work, a protein glycosylation (pgl) operon conferring prokaryotic N-glycosylation in C. jejuni JCM 2013 was cloned and identified. Fourteen open reading frames (ORFs) were found in the pgl operon. The operon organization was similar to that of C. jejuni NCTC 11168, with 98% and 99% identities in overall nucleotide sequence and amino acid sequence, respectively. The pgl operon was heterologously co-expressed with model protein CmeA in the Escherichia coli BL21 ΔwaaL mutant. The immuno- and lectin-blotting analysis indicated the protein glycosylation on the recombinant CmeA. In addition, to analyze the glycan composition, the recombinant CmeA was purified and subjected to in-gel trypsin digestion followed by mass spectrometry analysis. The mass spectrometry analysis showed the presence of the N-acetylhexosamine residue at the reducing end but not the predicted di-N-acetylbacillosamine (diNAcBac) residue. Further glycan structural study using the conventional fluorophore-labeling method revealed the GalNAcα-GalNAcα-(Hex-)HexNAc-HexNAc-HexNAc-HexNAc structure. Transcriptional analysis showed that UDP-diNAcBac synthases and diNAcBac transferase are transcribed but might not function in the constructed system. In conclusion, a pgl operon from C. jejuni JCM 2013 successfully functioned in E. coli, resulting in the observed prokaryotic glycosylation.
|
巻・号 |
118(3)
|
ページ |
256-62
|
公開日 |
2014-9-1
|
DOI |
10.1016/j.jbiosc.2014.02.011
|
PII |
S1389-1723(14)00063-2
|
PMID |
24650731
|
MeSH |
Acetylglucosamine / chemistry
Acetylglucosamine / metabolism
Amino Acid Sequence
Bacterial Proteins / chemistry
Bacterial Proteins / genetics*
Bacterial Proteins / metabolism
Base Sequence
Campylobacter jejuni / genetics*
Campylobacter jejuni / metabolism
Carbohydrate Sequence
Escherichia coli / genetics*
Escherichia coli / metabolism
Gene Expression Regulation, Bacterial*
Glycosylation
Humans
Molecular Sequence Data
Open Reading Frames
Operon*
Recombinant Proteins / chemistry
Recombinant Proteins / genetics
Recombinant Proteins / metabolism
Transcription, Genetic
|
IF |
2.366
|
引用数 |
3
|
WOS 分野
|
FOOD SCIENCE & TECHNOLOGY
BIOTECHNOLOGY & APPLIED MICROBIOLOGY
|
リソース情報 |
遺伝子材料 |
Genomic DNA of Campylobacter jejuni subsp. jejuni JCM 2013 (JGD08023) |
一般微生物 |
|