Human preproparathyroid hormone synthesized in Escherichia coli is transported to the surface of the bacterial inner membrane but not processed to the mature hormone.
cDNA encoding human preproPTH (hpreproPTH) was expressed in Escherichia coli to study the processing of the precursor to hPTH and its secretion by the bacterial secretory apparatus. We first constructed hybrid genes that differed randomly in the distance between the E. coli lac promoter's ribosomal binding site and DNA encoding a fusion protein with beta-galactosidase activity and the prepro sequence of hpreproPTH on the aminoterminus. Starting with clones identified as efficient producers of beta-galactosidase on indicator agar plates, the coding sequence for hpreproPTH was reconstituted intact. In a different construction we placed the hpreproPTH coding sequence downstream from the lac promoter at a distance of 12 base pairs from the ribosomal binding site. PTH immunoreactive proteins from multiple clones were identified by protein gel electrophoresis and by protein microsequencing. PTH-related proteins encoded by different plasmids were shown to be hpreproPTH with amino-terminal extensions of either two or four amino acids and as authentic hpreproPTH. Two hPTH fragments, hPTH(3-84) and hPTH(8-84), were also observed. The trypsin accessibility of hpreproPTH and of the two hPTH fragments in pulse-chase, cell-fractionation experiments using intact and lysed spheroplasts lets us conclude that the mammalian signal sequence directs hpreproPTH to the surface of the spheroplast membrane but is not appropriately cleaved by the signal peptidase.
Born
W
W
Endocrine Unit, Massachusetts General Hospital, Boston 02114.
Freeman
M
M
Hendy
G N
GN
Rapoport
A
A
Rich
A
A
Potts
J T
JT
Kronenberg
H M
HM
D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002462:Cell Membrane / Q000648:ultrastructure; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D008969:Molecular Sequence Data; D010281:Parathyroid Hormone / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics / Q000493:pharmacokinetics*; D010446:Peptide Fragments / Q000032:analysis; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics / Q000493:pharmacokinetics*; D014357:Trypsin / Q000494:pharmacology
D010281:Parathyroid Hormone; D010446:Peptide Fragments; D011498:Protein Precursors; C048158:parathyroid hormone (8-34); C054602:parathyroid hormone (8-84); C019056:preproparathormone; D014357:Trypsin