RRC ID 30956
Author Srichaisupakit A, Ohashi T, Fujiyama K.
Title Identification of a protein glycosylation operon from Campylobacter jejuni JCM 2013 and its heterologous expression in Escherichia coli.
Journal 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.
Volume 118(3)
Pages 256-62
Published 2014-9
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 2.015
Times Cited 2
WOS Category FOOD SCIENCE & TECHNOLOGY BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Resource
DNA material Genomic DNA of Campylobacter jejuni subsp. jejuni JCM 2013 (JGD08023)
General Microbes