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首页> 外文期刊>Biotechnology Progress >Novel secretion system of recombinant Saccharomyces cerevisiae using anN-terminus residue of human IL-1 beta as secretion enhancer
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Novel secretion system of recombinant Saccharomyces cerevisiae using anN-terminus residue of human IL-1 beta as secretion enhancer

机译：以人IL-1β的N端残基为分泌增强剂的新型酿酒酵母分泌系统

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摘要

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 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-1 beta, 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-alpha 1 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-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 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-1 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 hIL-1 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.
机译：人类白介素1 beta（hIL-1 beta）的N端序列被用作融合表达伴侣，以生产两种重组治疗蛋白，人类粒细胞集落刺激因子（hG-CSF）和人类生长激素（hGH） ，以酿酒酵母为宿主。表达盒包括乳酸克鲁维酵母的杀伤毒素的前导序列，成熟的hIL-1 beta的N端24个氨基酸（Ser5-Ala28），KEX2二元内肽酶切割位点和目标蛋白（hG-CSF或hGH） ）。基因表达受诱导的UAS（gal）/ MF-alpha 1启动子控制。利用上述表达载体，两种重组蛋白均被良好地分泌到具有高分泌效率的培养基中，并且特别地，重组hGH在培养液中积累至约1.3g / L。据推测这是由于融合的hIL-1β在酵母分泌途径中作为分泌增强剂的重要作用。在我们最近的报告中，各种免疫印迹分析表明，hIL-1β片段中驻留的核心N-糖基化可能对重组酿酒酵母中hG-CSF的高水平分泌至关重要。 。当向培养基中添加衣霉素后，N-糖基化被完全阻断时，尽管其他宿主来源的蛋白均被良好地分泌到培养液中，但hG-CSF和hGH的分泌降低到可以忽略的水平。衣霉素。纯化的hG-CSF的N端测序证实，融合蛋白从高尔基体中退出后，hIL-1β融合肽已被体内KEX2蛋白酶正确去除。根据本文提供的结果，强烈建议在设计用于分泌啤酒酵母中其他异源蛋白的表达系统中，hIL-1β肽的N末端融合可用作有效的分泌增强剂。 。

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