title: Expression, purification and characterization of low-glycosylation influenza neuraminidase in α-1,6-mannosyltransferase defective Pichia pastoris.
abstract: Influenza A viruses expose two major surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Although N-glycosylation is essential for many glycoproteins, the glycoproteins expressed in yeast are sometimes hyper-glycosylated, which maybe a primary hindrance to the exploitation of therapeutic glycoprotein production because glycoproteins decorated with yeast-specific glycans are immunogenic and show poor pharmacokinetic properties in humans. To elucidate the NA with different glycosylation in interaction with immunogenicity, here we reported the heterologous expression of influenza NA glycoprotein derived from influenza virus A/newCaledonia/20/99(H1N1) in wide-type Pichia pastoris, α-1,6-mannosyltransferase (och1)-defective P. pastoris and Escherichia coli. We also assessed the immunogenicity of hyper-glycosylated NA expressed in the wide-type, low-glycosylated NA expressed in och1-defective P. pastoris strain and non-glycosylated NA produced in E. coli. Recombinant NA was expressed in wide-type P. pastoris as a 59-97 above kDa glycoprotein, 52-57 kDa in the och1 defective strain, and as a 45 kDa non-glycoprotein in E. coli. The antibody titers of Balb/c mice were tested after the mice were immunized three times with 0.2, 1, or 3 μg purified recombinant NA. Our results demonstrated that after the second immunization, the antibody titer elicited with 1 μg low-glycosylated NA was 1:5,500, while it was 1:10 and 1:13 when elicited by 1 μg hyper-glycosylated and non-glycosylated NA. In the 0.2 μg dose groups, a high antibody titer (1:4,900) was only found after third immunization by low-glycosylated NA, respectively. These results suggest that low-glycosylation in och1-defective P. pastoris enhances the immunogenicity of recombinant NA and elicits similar antibody titers with less antigen when compared with hyper- and non-glycosylated NA. Thus, och1-defective P. pastoris may be a better yeast expression system for production of glycoproteins to research immunogenic characterization.
authors.affiliation: Department of Microorganism Engineering, Beijing Institute of Biotechnology, 20 Dongdajie Street, Fengtai District, Beijing 100071, China.
authors.forename: Yi-Li
authors.initials: YL
authors.lastname: Yang
authors.forename: Shao-Hong
authors.initials: SH
authors.lastname: Chang
authors.forename: Xin
authors.initials: X
authors.lastname: Gong
authors.forename: Jun
authors.initials: J
authors.lastname: Wu
authors.forename: Bo
authors.initials: B
authors.lastname: Liu
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000097:blood; D019149:Bioreactors; D015153:Blotting, Western; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli; D006031:Glycosylation; D053118:Influenza A Virus, H1N1 Subtype / Q000201:enzymology* / Q000235:genetics; D007252:Influenza Vaccines / Q000276:immunology* / Q000378:metabolism; D008364:Mannosyltransferases / Q000235:genetics; D008562:Membrane Glycoproteins / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009439:Neuraminidase / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: D000914:Antibodies, Viral; D017931:DNA Primers; D007252:Influenza Vaccines; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D008364:Mannosyltransferases; D009439:Neuraminidase