HIGH-LEVEL SECRETION OF CORRECTLY PROCESSED RECOMBINANT HUMAN INTERLEUKIN-1-BETA IN KLUYVEROMYCES-LACTIS

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 2-mu-like plasmid pKD1 from Kluyveromyces drosophilarum [Chen et al., Nucleic Acids Res. 14 (1986) 4471-4481] for the secretion of recombinant human interleukin-1-beta (reIL-1-beta). High levels of reIL-1-beta 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-1-beta, the 'pro'-sequence being derived from the human serum albumin-encoding gene, resulted in equally efficient secretion of mature IL-1-beta. Cytoplasmic production of Met-IL-1-beta, 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-1-beta, K. lactis reIL-1-beta is glycosylated. This glycosylation led to a 95% loss of its biological activity. Removal of the carbohydrate chains by endo-beta-N-acetylglucosamidase H treatment fully restored the biological activity. A modified form of IL-1-beta(Asn7 --> Gln7), in which the unique site for Asn-linked glycosylation was deleted, exhibited the same biological activity as native IL-1-beta. The level of secretion of mature recombinant IL-1-beta was glycosylation-independent.