N-Glycosylation of secretion enhancer peptide as influencing factor for the secretion of target proteins from Saccharomyces cerevisiae.
hIL-1beta-derived polypeptide, when fused to the N-terminal end of target proteins, exerts a potent secretion enhancer function in Saccharomyces cerevisiae. We investigated the effect of N-glycosylation of the secretion enhancer peptide on the secretion of target proteins. The N-terminal 24 amino acids (Ser5-Ala28) of human interleukin 1beta (hIL-1beta) and interleukin 1 receptor antagonist (IL-1ra) were used as secretion enhancer for synthesizing recombinant human granulocyte-colony stimulating factor (rhG-CSF) from S. cerevisiae. The mutation of potential N-glycosylation site, by substituting Gln for either Asn7 of N-terminal 24 amino acids of hIL-1beta (Asn7Gln) or Asn84 of IL-1ra (Asn84Gln), resulted in a dramatic reduction of rhG-CSF secretion efficiency. In contrast, the mutant containing an additional N-glycosylation site on the N-terminal 24 amino acids of hIL-1beta (Gln15Asn) secreted twice as much rhG-CSF into culture media as wild type hIL-1beta. These results show that N-glycosylation of the secretion enhancer peptide plays an important role in increasing the secretion efficiency of the downstream target proteins. The results also suggest that judicious choice of enhancer peptide and the control of its glycosylation could be of general utility for secretory production of heterologous proteins from S. cerevisiae.
Han
Kyoung Sim
KS
Protheon Incorporated, Yonsei Engineering Research Center B120E, Seodaemun-Gu, Seoul, Republic of Korea.
Kim
Seo Il
SI
Choi
Seong Il
SI
Seong
Baik Lin
BL
D015536:Down-Regulation; D006031:Glycosylation; D016179:Granulocyte Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007375:Interleukin-1 / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017472:Receptors, Interleukin-1 / Q000037:antagonists & inhibitors / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors
D007375:Interleukin-1; D010455:Peptides; D017472:Receptors, Interleukin-1; D011993:Recombinant Fusion Proteins; D016179:Granulocyte Colony-Stimulating Factor