Characterization of a K26Q site-directed mutant of human parathyroid hormone expressed in yeast.
Human parathyroid hormone (hPTH) is susceptible to proteolytical cleavage both in humans and when expressed as a secretory product in Escherichia coli (Høgseth, A., Blingsmo, O. R., Saether, O., Gautvik, V. T., Holmgren, E., Hartmanis, M., Josephson, S., Gabrielsen, O. S., Gordeladze, J. O., Alestrøm, P., and Gautvik, K. M. (1990) J. Biol. Chem. 265, 7338-7344) and Saccharomyces cerevisiae (Gabrielsen, O. S., Reppe, S., Saether, O., Blingsmo, O. R., Sletten, K., Gordeladze, J. O., Høgset, A., Gautvik, V. T., Alestrøm, P., Oyen, T. B., and Gautvik, K. M. (1990) Gene (Amst.) 90, 255-262). In the latter system, one major site of cleavage was identified (Arg25-Lys26 decreased Lys27). To produce hPTH resistant to this proteolytic processing, a point mutation changing Lys26 to Gln was introduced, and the modified gene expressed in S. cerevisiae as a fusion protein with the alpha-factor leader sequence. The resulting major form of hPTH secreted to the growth medium was of full length showing that the mutation had eliminated internal processing. Consequently, the yield of the mutant hormone was significantly higher than obtained with the natural peptide. Using improved purification procedures, a significantly higher purity was also obtained. The secreted mutant hPTH-(1-84,Q26) had the correct size, full immunological reactivity with two different hPTH antisera, correct amino acid composition and N-terminal sequence, and correct mass as determined by mass spectrometry. Furthermore, the introduced mutation did not reduce the biological activity of the hormone as judged from its action in three biological assay systems: 1) a hormone-sensitive osteoblast adenylate cyclase assay; 2) an in vivo calcium mobilizing assay in rats; and 3) an in vitro bone resorption assay.
Reppe
S
S
Department of Biochemistry, University of Oslo, Norway.
Gabrielsen
O S
OS
Olstad
O K
OK
Morrison
N
N
Saether
O
O
Blingsmo
O R
OR
Gautvik
V T
VT
Gordeladze
J
J
Haflan
A K
AK
Voelkel
E F
EF
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005800:Genes, Fungal; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010281:Parathyroid Hormone / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D010281:Parathyroid Hormone