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The influence of N‐glycosylation and C‐terminal sequence on secretion of HBV large surface antigen from S. cerevisiae
Author(s) -
Park JinSeung,
Seo HyukSeong,
Yum JungSun,
Moon Hong Mo,
Lee Jeewon
Publication year - 2005
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.20640
Subject(s) - degree (music) , degree of polymerization , glycosylation , first degree relatives , chemistry , microbiology and biotechnology , biology , biochemistry , medicine , physics , organic chemistry , radiology , acoustics , polymerization , family history , polymer
In Saccharomyces cerevisiae , we synthesized and secreted L‐HBVsAg (named as pre‐S(Met1 to Asn174)::S(Met175 to Ile400)) and three mutants, i.e., pre‐S°°::S (Asn15Gln and Asn123Gln), pre‐S°°::S° (Asn15Gln, Asn123Gln, and Asn320Gln), and pre‐S°°::S°° (Asn15Gln, Asn123Gln, Asn233Gln, and Asn320Gln). All of the secreted pre‐S::S was N‐glycosylated, i.e., hyper‐mannosylated. In the secretion of pre‐S°°::S and pre‐S°°::S°, besides the hyper‐mannosylated form, another immunoreactive protein with much lower molecular mass was observed, which seems to be unglycosylated form of pre‐S°°::S and pre‐S°°::S°. Only a part of the secreted pre‐S°°::S or pre‐S°°::S° molecules was N‐glycosylated, and the site for the partial N‐glycosylation seems to be Asn233 in S‐antigen region. Compared to the N‐glycosylated pre‐S°°::S and pre‐S°°::S°, pre‐S°°::S°° (non‐N‐glycosylated mutant) was secreted with lower secretion efficiency but showed apparent immunoreactivity to anti‐S antigen monoclonal Ab. Interestingly, unlike pre‐S°°::S°° with authentic C‐terminus, the recombinant pre‐S°°::S°° with C‐terminal myc or poly‐histidine tag (pre‐S°°::S°°::tag) was almost all aggregated into insoluble proteins in the intracellular region. Conclusively, the C‐terminal sequence and glycosylation in S‐antigen region seem to be of crucial importance in determining the secretion efficiency of L‐HBVsAg in S. cerevisiae . © 2005 Wiley Periodicals, Inc.