Novel Secretion System of Recombinant Saccharomyces cerevisiae Using an N‐terminus Residue of Human IL‐1β as Secretion Enhancer

Abstract

An N-terminus sequence of human interleukin 1beta (hIL-1beta) 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 cerevisiae as a host. The expression cassette comprised the leader sequence of killer toxin of Kluyveromyces lactis, the N-terminus 24 amino acids (Ser5-Ala28) of mature hIL-1beta, the KEX2 dibasic endopeptidase cleavage site, and the target protein (hG-CSF or hGH). The gene expression was controlled by the inducible UAS(gal)/MF-alpha1 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 of fused hIL-1beta 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-1beta fragment is likely to be of crucial importance in the high-level secretion of hG-CSF from the recombinant S. cerevisiae. When the N-glycosylation was completely blocked with 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 proteins were well secreted to the culture broth regardless of the presence of tunicamycin. The N-terminal sequencing of the purified hG-CSF verified that the hIL-1beta 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 hIL-1beta 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.

Recombinant enterokinase light chain with affinity tag: Expression from Saccharomyces cerevisiae and its utilities in fusion protein technology

“…The cDNA encoding 235 residues of EK L (26.3 kDa) was cloned into SecHancer vector pIL20GH to yield pIL20EK L . EK gene is linked in frame with Nterminal 24 residues of human interleukin-1b as secretion enhancer after the killer toxin leader sequence (Lee et al, 1999). A KEX2 protease cleavage site is located between the above secretion enhancer and EK L .…”

“…cerivisiae 2805 (pep4:: H1S3, pro1-d, can1, Gal2, his3d, ura3-52) was transformed with the above plasmids. The yeast cells were cultured as previously described (Lee et al, 1999) in 10 mL of SD Ura media [0.8 g/L Complete Supplement Media (BIO101), 6.7 g/L yeast nitrogen base without amino acid (DIFCO), 2% glucose] and incubated at 30°C overnight at 250 rpm. The culture broth was centrifuged, and the expression was induced by suspending cell pellet in YPGa1 media (1% yeast extract, 2% Bacto-peptone, 2% galactose).…”

“…The combination of conventional secretion leader and the enhancer peptide greatly increased the secretion level of various proteins (Lee et al, 1999). Using this secretion enhancer system, we expressed the catalytic subunit of enterokinase, EK L .…”

Identification and Use of Zinc Finger Transcription Factors That Increase Production of Recombinant Proteins in Yeast and Mammalian Cells

“…Filter circles (82 mm) of 0.45 µm nitrocellulose, agarose type IV, and the hGH ELISA kit were purchased from Millipore (U.S.A.), Sigma Chemical (U.S.A.), and Roche (Germany), respectively. The hGH expression plasmid, pktXGH, was described previously (16). The expression cassette in pktXGH comprised the leader sequence of killer toxin of Kluyveromyces lactis, the KEX2 dibasic endopeptidase cleavage site, and the hGH gene.…”

“…In S. cerevisiae, several "super-secreting" or "over-secreting" mutants have been isolated, and these yeast strains yield high concentrations of secreted heterologous proteins (2,3). An alternative approach has been to isolate several signal sequences from yeast secretory proteins, fuse them to the heterologous proteins, express the fusion proteins in yeast cells, and isolate the secreted recombinant proteins from the yeast cell media (11)(12)(13)(14)(15)(16). In mammalian cells, amplification of the genes that encode recombinant proteins of interest is a key method used to enhance the productivity of these recombinant products (17,18).…”