Nomura N, Yamada H, Matsubara N, Horinouchi S, Beppu T
Department of Biotechnology, University of Tokyo, Japan.
Appl Microbiol Biotechnol. 1995 Mar;42(6):865-70. doi: 10.1007/BF00191183.
As the first step for production of rat apolipoprotein E (rApoE) in Saccharomyces cerevisiae, the rApoE cDNA was cloned and its nucleotide sequence was determined. When the intact rApoE gene including the presequence-encoding region was expressed under the control of the yeast GAL7 promoter, no protein immunoreactive with anti-rApoE antibody was detected either in the culture medium or inside the cells. For the purpose of the extracellular production of rApoE, three fusion genes were constructed in which the mature rApoE-encoding sequence was connected after the pre, prepro, and whole regions of the gene encoding a fungal aspartic proteinase, Mucor pusillus rennin (MPP), since MPP is efficiently secreted from recombinant S. cerevisiae containing the MPP gene. When these three fusion genes were expressed under the control of the GAL7 promoter, only one, encoding the mature rApoE connected to the whole MPP sequence, directed efficient secretion of the fused protein. The maximum yield of the fused protein secreted into the medium reached 11.8 mg/l and the calculated rApoE part was 5.3 mg in the fused protein. The excreted fusion protein was glycosylated at the original two sites in the MPP part. The fused protein was gradually degraded in the medium probably by proteases of the host cell, because no such degradation occurred in a yeast pep4mutant strain.
作为在酿酒酵母中生产大鼠载脂蛋白E（rApoE）的第一步，克隆了rApoE cDNA并测定了其核苷酸序列。当包含前序列编码区的完整rApoE基因在酵母GAL7启动子的控制下表达时，在培养基或细胞内均未检测到与抗rApoE抗体发生免疫反应的蛋白质。为了在细胞外生产rApoE，构建了三个融合基因，其中成熟的rApoE编码序列连接在编码真菌天冬氨酸蛋白酶毛霉凝乳蛋白酶（MPP）的基因的前导区、前原区和整个区域之后，因为MPP能从含有MPP基因的重组酿酒酵母中有效分泌。当这三个融合基因在GAL7启动子的控制下表达时，只有一个编码连接到整个MPP序列的成熟rApoE的融合基因能指导融合蛋白的有效分泌。分泌到培养基中的融合蛋白的最大产量达到11.8 mg/l，融合蛋白中计算得出的rApoE部分为5.3 mg。分泌的融合蛋白在MPP部分的原始两个位点进行了糖基化。融合蛋白在培养基中可能被宿主细胞的蛋白酶逐渐降解，因为在酵母pep4突变株中没有发生这种降解。
