Fleer R, Chen X J, Amellal N, Yeh P, Fournier A, Guinet F, Gault N, Faucher D, Folliard F, Fukuhara H
Rhône Poulenc Rorer, Biotechnology, Department, Vitry, France.
Gene. 1991 Nov 15;107(2):285-95. doi: 10.1016/0378-1119(91)90329-a.
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.
乳糖同化酵母乳酸克鲁维酵母已被开发成为用于合成和分泌人蛋白质的微生物宿主。在此，我们报道了基于果蝇克鲁维酵母的2μm类质粒pKD1 [Chen等人，《核酸研究》14 (1986) 4471 - 4481] 的多拷贝载体用于分泌重组人白细胞介素-1β（reIL-1β）。当结构基因与源自乳酸克鲁维酵母杀伤毒素“前”区的合成分泌信号框内融合时，高水平的reIL-1β被分泌到生长培养基中。对分泌蛋白的N端测序显示信号序列具有高效（大于95%）的成熟。以前原白细胞介素-1β形式合成，“原”序列源自人血清白蛋白编码基因，同样能高效分泌成熟的白细胞介素-1β。发现无分泌信号的甲硫氨酸白细胞介素-1β在细胞质中的产生对乳酸克鲁维酵母有毒。与酿酒酵母一样 [Baldari等人，《欧洲分子生物学组织杂志》6 (1987) 229 - 234]，但与天然人白细胞介素-1β不同，乳酸克鲁维酵母reIL-1β是糖基化的。这种糖基化导致其生物学活性丧失95%。通过内切β-N-乙酰葡糖胺糖苷酶H处理去除糖链可完全恢复生物学活性。一种修饰形式的白细胞介素-1β（Asn7→Gln7）删除了天冬酰胺连接糖基化的独特位点，其生物学活性与天然白细胞介素-1β相同。成熟重组白细胞介素-1β的分泌水平与糖基化无关。
