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.
authors.affiliation: Department of Chemical and Biological Engineering, Korea University, Anam-Dong, Sungbuk-Ku, Seoul 136-701, Republic of Korea. leejw@korea.ac.kr
authors.forename: Jeewon
authors.initials: J
authors.lastname: Lee
authors.forename: Jin-Seung
authors.initials: JS
authors.lastname: Park
authors.forename: Je-Young
authors.initials: JY
authors.lastname: Moon
authors.forename: Ki-Yong
authors.initials: KY
authors.lastname: Kim
authors.forename: Hong-Mo
authors.initials: HM
authors.lastname: Moon
mesh_terms: D019943:Amino Acid Substitution; D003001:Cloning, Molecular; D004355:Drug Stability; D006031:Glycosylation; D006514:Hepatitis B Surface Antigens / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007375:Interleukin-1 / Q000235:genetics; D016254:Mutagenesis, Insertional; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D006514:Hepatitis B Surface Antigens; D007375:Interleukin-1; C077433:Pre-S protein, Duck hepatitis B virus; D011498:Protein Precursors; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; C050806:presurface protein 1, hepatitis B surface antigen; C050807:presurface protein 2, hepatitis B surface antigen