title: Enhanced accumulation of secreted human growth hormone by transgenic tobacco cells correlates with the introduction of an N-glycosylation site.
abstract: Extracellular secretion of recombinant proteins from plant cell suspension culture will simplify the protein purification procedure and greatly reduce the production cost. Our early work indicated that presence of hydroxyproline-O-glycosylation at the C- or N-terminus of the target protein boosted the secreted yields in the culture medium. Inspired by early successes, we tested the possibility of introducing an N-glycosylation site to facilitate the secretion of human growth hormone (hGH) from cultured tobacco cells. Three N-glycosylated hGH fusion proteins, designated NAS-EK-hGH, NAS-Kex2-hGH and hGH-NAS, were expressed in tobacco BY-2 cells. Where NAS denotes the "Asn-Ala-Ser" consensus sequence for N-glycosylation; EK denotes an enterokinase cleavage site and Kex2 a sequence to be cleaved by a Golgi-localized Kex2p-like protease. Our results indicated that a single N-glycan attached either at the N-terminus or C-terminus of hGH correlated with enhanced extracellular accumulation of the transgenic proteins; the secreted yield of NAS-EK-hGH and hGH-NAS was 70-90 fold greater than the control targeted, non-glycosylated hGH. NAS-Kex2-hGH was subject to partial cleavage of the N-glycan tag at the Kex2 site in Golgi apparatus, and therefore gave lower yields than the other two constructs.
authors.affiliation: Department of Chemistry and Biochemistry, Ohio University, Athens, United States. jxu@astate.edu
authors.forename: Jianfeng
authors.initials: J
authors.lastname: Xu
authors.forename: Marcia
authors.initials: M
authors.lastname: Kieliszewski
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002241:Carbohydrates / Q000032:analysis; D002478:Cells, Cultured; D003470:Culture Media / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000378:metabolism; D006031:Glycosylation / Q000187:drug effects; D019382:Human Growth Hormone / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D020539:Sequence Analysis, Protein; D014026:Nicotiana / Q000166:cytology* / Q000187:drug effects / Q000235:genetics*; D014415:Tunicamycin / Q000494:pharmacology
chemical_list: D002241:Carbohydrates; D003470:Culture Media; D006023:Glycoproteins; D011994:Recombinant Proteins; D014415:Tunicamycin; D019382:Human Growth Hormone