High-level secretion of correctly processed recombinant human interleukin-1β in Kluyveromyces lactis

Abstract The lactose-assimilating yeast, Kluyveromyces lactis , has been developed as a microbial host for the synthesis and secretion of human proteins. Here, we report the use of multi-copy vectors based on the 2μ-like plasmid pKD1 from Kluyveromyces drosophilarum [Chen et al., Nucleic Acids Res, 14 (1986) 4471–4481] for the secretion of recombinant human interleukin-1β (reIL-1β). High levels of reIL-1β were secreted into the growth medium when the structural gene was fused in-frame to a synthetic secretion signal derived from the ‘pre’-region of the K. lactis killer toxin. N-terminal sequencing of the excreted protein showed highly efficient (> 95%) maturation of the signal sequence. Synthesis as prepro-IL-1β, the ‘pro’-sequence being derived from the human serum albumin-encoding gene, resulted in equally efficient secretion of mature IL-1β Cytoplasmic production of Met-IL-1β, without a secretion signal, was found to be toxic to K. lactis . As in Saccharomyces cerevisiae [Baldari et al, EMBO J. 6 (1987) 229–234], but unlike native human IL-1β K. lactis reIL-1β is glycosylated. This glycosylation led to a 95% loss of its biological activity. Removal of the carbohydrate chains by endo-β- N -acetyl-glucosamidase H treatment fully restored the biological activity. A modified form of IL-1β (Asn 7 → Gln 7 ), in which the unique site for Asn-linked glycosylation was deleted, exhibited the same biological activity as native IL-1β. The level of secretion of mature recombinant IL-1β was glycosylation-independent.