Novel secretion system of recombinant Saccharomyces cerevisiae using an N-terminus residue of human IL-1 beta as secretion enhancer

An N-terminus sequence of human interleukin 1 beta (hIL-1 beta) was used as a fusion expression partner for the production of two recombinant therapeu tic proteins, human granulocyte-colony stimulating factor (hG-CSF) and huma n growth hormone (hGH), using Saccharomyces cerevisiae as a host. The expre ssion cassette comprised the leader sequence of killer toxin of Kluyveromyc es lactis, the N-terminus 24 amino acids (Ser5-Ala28) of mature hIL-1 beta, the KEX2 dibasic endopeptidase cleavage site, and the target protein (hG-C SF or hGH). The gene expression was controlled by the inducible UAS(gal)/MF -alpha 1 promoter. With the expression vector above, both recombinant prote ins were well secreted into culture medium with high secretion efficiencies , and especially, the recombinant hGH was accumulated up to around 1.3 g/L in the culture broth. This is due presumably to the significant role of fus ed hIL-1 beta as secretion enhancer in the yeast secretory pathway. In our recent report, various immunoblotting analyses have shown that the presence of a core N-glycosylation resident in the hIL-1 beta fragment is likely to be of crucial importance in the high-level secretion of hG-CSF from the re combinant S. cerevisiae. When the N-glycosylation was completely blocked wi th the addition of tunicamycin to the culture, the secretion of hG-CSF and hGH was decreased to a negligible level although the other host-derived pro teins were well secreted to the culture broth regardless of the presence of tunicamycin. The N-terminal sequencing of the purified hG-CSF verified tha t the hIL-1 beta fusion peptide was correctly removed by in vivo KEX2 prote ase upon the exit of fusion protein from Golgi complex. From the results pr esented in this article, it is strongly suggested that the N-terminus fusio n of the hIL-1 beta peptide could be utilized as a potent secretion enhance r in the expression systems designed for the secretory production of other heterologous proteins from S. cerevisiae.