Sakai A, Ozawa F, Higashizaki T, Shimizu Y, Hishinuma F
Laboratory of Molecular Genetics, Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
Biotechnology (N Y). 1991 Dec;9(12):1382-5. doi: 10.1038/nbt1291-1382.
We have designed an advanced delta-integration system (integration of genes into the delta-sequence of yeast retrotransposon Ty) and used it for secretion of human nerve growth factor (hNGF) from Saccharomyces cerevisiae. The expression and secretion of hNGF was directed by the PGK promoter and MF alpha 1 prepro-signal. Using two selectable markers (URA3 and leu2-d), haploid yeast strains were constructed with approximately 20 copies of a delta-integrated hNGF expression cassette on four chromosomes. The strain secreted hNGF at levels 3-4 fold higher than a 2 micron-based plasmid. Northern and Western analyses revealed that the oversecretion was caused by an increased amount of mRNA. We also detected an unusual processing of the MF alpha 1 prepro-hNGF fusion protein that required the pep4 mutation. Application of this system for industrial purposes is discussed.
我们设计了一种先进的δ-整合系统（将基因整合到酵母逆转录转座子Ty的δ序列中），并将其用于酿酒酵母分泌人神经生长因子（hNGF）。hNGF的表达和分泌由PGK启动子和MFα1前原信号引导。利用两个选择标记（URA3和leu2-d），构建了单倍体酵母菌株，在四条染色体上有大约20个拷贝的δ-整合hNGF表达盒。该菌株分泌hNGF的水平比基于2μm质粒的菌株高3至4倍。Northern和Western分析表明，过量分泌是由mRNA量增加引起的。我们还检测到MFα1前原-hNGF融合蛋白的一种异常加工，这需要pep4突变。讨论了该系统在工业上的应用。
