High-level production of human parathyroid hormone (hPTH) by induced expression in Saccharomyces cerevisiae.
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.
Vad
R
R
Department of Biochemistry, University of Oslo, Oslo, N-0316, Norway.
Moe
E
E
Saga
K
K
Kvinnsland
A M
AM
Oyen
T B
TB
D003001:Cloning, Molecular; D003300:Copper / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D010281:Parathyroid Hormone / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013696:Temperature
D010281:Parathyroid Hormone; D011994:Recombinant Proteins; D003300:Copper