Vad R, Moe E, Saga K, Kvinnsland A M, Oyen T B
Department of Biochemistry, University of Oslo, Oslo, N-0316, Norway.
Protein Expr Purif. 1998 Aug;13(3):396-402. doi: 10.1006/prep.1998.0912.
Saccharomyces cerevisiae was used as host for high-level production of intact human parathyroid hormone (hPTH). The yield increased about 30-fold by changing from the constitutive MFalpha promoter to the inducible CUP1 promoter in the expression cassettes, use of another host strain, and optimization of growth conditions where especially the pH value was crucial. The secreted products consisted mainly of intact hormone, hPTH(1-84). In addition, two C-terminally truncated forms that lacked the four or five last amino acid residues, hPTH(1-80) and hPTH(1-79), were identified. These hPTH forms migrated aberrantly by SDS-PAGE as 14-kDa proteins, while the real masses measured by mass spectrometry on HPLC-purified products were about 9 kDa. Availability of such easily purified truncated forms will be valuable for studies of how the C-terminal residues affect the structure and function of the hormone. Combination of mutations and disruptions of the host genes encoding proteinase A, B, carboxypeptidase Y, and Kex1p or Mkc7p did not influence the C-terminal deletions. The secretion of hPTH could be enhanced by overexpression of the yeast syntaxin gene SSO2, but the total level of the hormone was not improved due to impaired growth.
酿酒酵母被用作宿主，用于高水平生产完整的人甲状旁腺激素（hPTH）。通过在表达盒中从组成型MFalpha启动子转换为诱导型CUP1启动子、使用另一种宿主菌株以及优化生长条件（其中pH值尤为关键），产量提高了约30倍。分泌产物主要由完整的激素hPTH(1 - 84)组成。此外，还鉴定出了两种C末端截短形式，它们缺少最后四个或五个氨基酸残基，即hPTH(1 - 80)和hPTH(1 - 79)。这些hPTH形式在SDS-PAGE中以14 kDa蛋白的形式异常迁移，而通过质谱法对HPLC纯化产物测量的实际质量约为9 kDa。这种易于纯化的截短形式的可得性对于研究C末端残基如何影响激素的结构和功能将是有价值的。对编码蛋白酶A、B、羧肽酶Y以及Kex1p或Mkc7p的宿主基因进行突变和破坏的组合并不影响C末端缺失。酵母 syntaxin 基因SSO2的过表达可以增强hPTH的分泌，但由于生长受损，激素的总水平并未提高。
