Efficient secretion of human parathyroid hormone by Saccharomyces cerevisiae.
A cDNA encoding mature human parathyroid hormone (hPTH) was expressed in Saccharomyces cerevisiae, after fusion to the prepro region of yeast mating factor alpha (MF alpha). Radioimmunoassay showed high levels of hPTH immunoreactive material in the growth medium (up to 10 micrograms/ml). More than 95% of the immunoreactive material was found extracellularly as multiple forms of hormone peptides. Three internal cleavage sites were identified in the hPTH molecule. The major cleavage site, after a pair of basic amino acids (aa) (Arg25Lys26 decreases Lys27), resembles that recognized by the KEX2 gene product on which the MF alpha expression-secretion system depends. The use of a protease-deficient yeast strain and the addition of high concentrations of aa to the growth medium, however, not only changed the peptide pattern, but also resulted in a significant increase in the yield of intact hPTH (1-84) (more than 20% of the total amount of immunoreactive material). The secreted hPTH (1-84) migrates like a hPTH standard in two different gel-electrophoretic systems, co-elutes with standard hPTH on reverse-phase high-performance liquid chromatography, reacts with two hPTH antibodies raised against different parts of the peptide, has a correct N-terminal aa sequence, and has full biological activity in a hormone-sensitive osteoblast adenylate cyclase assay.
Gabrielsen
O S
OS
Department of Biochemistry, University of Oslo, Norway.
Reppe
S
S
Saether
O
O
Blingsmo
O R
OR
Sletten
K
K
Gordeladze
J O
JO
Høgset
A
A
Gautvik
V T
VT
Alestrøm
P
P
Oyen
T B
TB
D000262:Adenylyl Cyclases; D000595:Amino Acid Sequence; D001483:Base Sequence; D015870:Gene Expression; D006801:Humans; D000072235:Mating Factor; D008969:Molecular Sequence Data; D009154:Mutation; D010281:Parathyroid Hormone / Q000235:genetics* / Q000378:metabolism; D010455:Peptides / Q000235:genetics; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D014170:Transformation, Genetic
D010281:Parathyroid Hormone; D010455:Peptides; D011994:Recombinant Proteins; D000072235:Mating Factor; D000262:Adenylyl Cyclases