Hiramatsu R, Yamashita T, Aikawa J, Horinouchi S, Beppu T
Department of Agricultural Chemistry, Faculty of Agriculture, University of Tokyo, Japan.
Appl Environ Microbiol. 1990 Jul;56(7):2125-32. doi: 10.1128/aem.56.7.2125-2132.1990.
An aspartic proteinase, Mucor pusillus rennin (MPR), of filamentous fungus Mucor pusillus, is efficiently secreted from a transformant of Saccharomyces cerevisiae containing the intact MPR gene. To test the usefulness of the MPR leader peptide in secretion of heterologous proteins from yeast cells, several plasmids encoding the fusion proteins composed of different parts of the NH2-terminal region of prepro-MPR and human growth hormone (hGH) were constructed. The parts of the leader peptide upstream of hGH were the whole prepro-peptide following the NH2-terminal region of mature MPR in JGH1, the intact pre-sequence and a part of the pro-sequence in JGH2, and the putative signal sequences of the NH2-terminal 18 and 22 amino acids in JGH3 and JGH7, respectively. When the hGH genes fused to these leader sequences were expressed in yeast cells under the control of the yeast GAL7 promoter, proteins of various sizes immunoreactive with the anti-hGH antibody were secreted into the medium. Among the plasmids mentioned above, JGH2 directed the greatest secretion of the protein of 23 kilodaltons in size, which contained the expected NH2-terminal amino acid sequence of an additional eight amino acids derived from the pro-peptide of MPR. The addition of the GAL10 terminator downstream of the hGH gene in JGH2 resulted in a greater than three- to fivefold increase in the secretion, whereas the insertion of the GAL4 gene, which is a positive regulator for the GAL system, had no significant effect. The improved yield of the total protein of hGH secreted into the medium reached approximately 10 mg/liter.
丝状真菌米根霉的一种天冬氨酸蛋白酶——米根霉凝乳酶（MPR），可从含有完整MPR基因的酿酒酵母转化体中高效分泌。为了测试MPR前导肽在酵母细胞分泌异源蛋白中的作用，构建了几种质粒，这些质粒编码由前体MPR氨基末端区域的不同部分与人生长激素（hGH）组成的融合蛋白。hGH上游的前导肽部分在JGH1中是成熟MPR氨基末端区域之后的整个前体肽，在JGH2中是完整的前序列和部分前肽序列，在JGH3和JGH7中分别是氨基末端18和22个氨基酸的推定信号序列。当与这些前导序列融合的hGH基因在酵母GAL7启动子的控制下在酵母细胞中表达时，各种大小的与抗hGH抗体免疫反应的蛋白被分泌到培养基中。在上述质粒中，JGH2指导分泌的23千道尔顿大小的蛋白最多，该蛋白包含来自MPR前肽的另外八个氨基酸的预期氨基末端氨基酸序列。在JGH2的hGH基因下游添加GAL10终止子导致分泌增加超过三到五倍，而插入作为GAL系统正调控因子的GAL4基因没有显著影响。分泌到培养基中的hGH总蛋白产量提高到约10毫克/升。
