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 (greater than 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-acetyl-glucosamidase 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 ws glycosylation-independent.
Fleer
R
R
Rhône Poulenc Rorer, Biotechnology, Department, Vitry, France.
Chen
X J
XJ
Amellal
N
N
Yeh
P
P
Fournier
A
A
Guinet
F
F
Gault
N
N
Faucher
D
D
Folliard
F
F
Fukuhara
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D015870:Gene Expression / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D007375:Interleukin-1 / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D055646:Killer Factors, Yeast; D007700:Kinetics; D007716:Kluyveromyces / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009183:Mycotoxins / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011499:Protein Processing, Post-Translational / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000378:metabolism
D007375:Interleukin-1; D055646:Killer Factors, Yeast; D009183:Mycotoxins; D011993:Recombinant Fusion Proteins; C058975:zymocin