Novel Secretion System of Recombinant Saccharomyces cerevisiae Using an N-terminus Residue of Human IL-1(beta)as secretion Enhancci

An N-terminus sequence of human mterleukin 1(beta)(hIL-1(beta)was used as a fusion expression partner for the production of two recombinant therapeutic proteins, human granulocyte-colony stimulating factor (hG-CSF) and human growth hormone (hGH), using Saccharomyces cereuisiae as a host. The expression cassette comprised the leader sequence of killer toxin of Kluyueromyces lactis, the N-terminus 24 amino acids (Ser5la28) of mature hIL-1(beta), the KEX2 dibasic endopeptidase cleavage site, and the target protein (hG-CSF or hGH). The gene expression was controlled by the inducible UASgai/ MF-(xl promoter. With the expression vector above, both recombinant proteins 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 offused hlL-l(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 hlL-l(beta)fragment is likely to be of crucial importance in the high-level secretion of hG-CSF from the recombinant S. cereuisiae. When the N-glycosylation was completely blocked with the addition oftunicamycin to the culture, the secretion of hG-CSF and hGH was decreased to a negligible level although the other host-derived proteins were well secreted to the culture broth regardless of the presence oftunicamycin. The N-terminal sequencing of the purified hG-CSF verified that the hlL-l(beta)fusion peptide was correctly removed by in vivo KEX2 protease upon the exit of fusion protein from Golgi complex. From the results presented in this article, it is strongly suggested that the N-terminus fusion of the hlL-l(beta)peptide could be utilized as a potent secretion enhancer in the expression systems designed for the secretory production of other heterologous proteins from S. cerevisiae.