Title : The influence of glycosylation on secretion, stability, and immunogenicity of recombinant
HBV pre-S antigen synthesized in Saccharomyces cerevisiae
Abstract :
- Three types of recombinant pre-S antigens (i.e., pre-S1S2) of hepatitis B virus (HBV) were synthesized in Saccharomyces cerevisiae and secreted into extracellular medium: wild type (pre-S1S2) and two mutant antigens, pre-S1 degrees S2 (Asn15Gln) and pre-S1 degrees S2 degrees (Asn15Gln and Asn123Gln)
- An N-terminus sequence (Ser5-Ala28) of human interleukin 1 beta ( hIL-1 beta ) was used as synthetic prosequence of recombinant HBV surface antigen ( pre-S ), secreted from S. cerevisiae
- The expression cassette comprised the signal peptide of the killer toxin of Kluyveromyces lactis, the synthetic prosequence above, KEX2 dibasic endopeptidase cleavage site ( -Lys-Arg- ), and the surface antigen
- The recombinant pre-S1S2 and pre-S1 degrees S2 were secreted in the hyper-mannosylated form, while the recombinant pre-S1 degrees S2 degrees was produced without N-glycosylation
- It has been demonstrated that the two particular N-linked glycans at Asn15 and Asn123 interfered with the B-cell response to the HBV-derived pre-S1S2, resulting in low titers of pre-S1S2-neutralizing antibodies
- This problem was overcome by eliminating both of the N-glycosylation signals
- Despite enhanced immunogenicity, the recombinant pre-S1 degrees S2 degrees showed two major problems: (1) inefficient Kex2 cleavage process in the secretory pathway and (2) the severe proteolytic degradation by yeast proteases
- The efficiency of Kex2 cleavage increased dramatically by removing N-glycosylation signal in the synthetic prosequence, but the proteolysis of pre-S1 degrees S2 degrees was somewhat inevitable
- Further systematic approaches including modulation of degree of N-glycosylation or relocation of N-glycosylation sites in the recombinant pre-S1S2 may make it possible to achieve both enhanced immunogenicity and resistance towards proteolytic degradation of the secreted pre-S antigen
Output (sent_index, trigger,
protein,
sugar,
site):
- 2. used, , -, -, sequence
- 5. Asn123, , -, the two particular N-linked glycans, Asn15 and Asn123
- 5. Asn15, , -, the two particular N-linked glycans, Asn15 and Asn123
- 9. N-glycosylation, , -, -, sites
Output(Part-Of) (sent_index,
protein,
site):
- 2. interleukin 1 beta, sequence
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):
- 5. HBV pre-S, the two particular N-linked glycans, Asn15 and Asn123