Purification and characterization of recombinant human parathyroid hormone-related protein.
Full-length human parathyroid hormone-related protein (PTHrP-(1-141] as well as a carboxyl-terminal shortened form (PTHrP-(1-108] have been expressed from recombinant DNA-derived clones. These proteins were expressed in Escherichia coli as fusion proteins so that cyanogen bromide cleavage yields the desired product. Both proteins were purified and then characterized by sodium dodecyl sulfate gel electrophoresis, amino-terminal amino acid sequencing, peptide mapping, and mass spectral analysis. Recombinant PTHrP-(1-141), PTHrP-(1-108), synthetic PTHrP-(1-34), and naturally derived PTHrP are all equipotent in the stimulation of cyclic AMP levels in the osteoblast-like cell line UMR 106-01. However, PTHrP-(1-141) and -(1-108) are two to four times more active than PTHrP-(1-34) in the stimulation of plasminogen activator activity from this cell line. PTHrP-(1-141) reacts equipotently with PTHrP-(1-34) in a radioimmunoassay using an antiserum prepared against PTHrP-(1-34). PTHrP-(1-141), -(1-108), and -(1-84) were used as PTHrP-specific mobility standards on sodium dodecyl sulfate gel electrophoresis to determine the approximate length of two forms of naturally derived PTHrP. The data show that PTHrP purified from the lung tumor cell line BEN contains a major form of about 108 amino acids and another form of about 141 amino acids.
Hammonds
R G
RG
Department of Developmental Biology, Genentech, Inc., South San Francisco, California 94080.
McKay
P
P
Winslow
G A
GA
Diefenbach-Jagger
H
H
Grill
V
V
Glatz
J
J
Rodda
C P
CP
Moseley
J M
JM
Wood
W I
WI
Martin
T J
TJ
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000096:biosynthesis; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000302:isolation & purification* / Q000502:physiology; D012516:Osteosarcoma; D010281:Parathyroid Hormone / Q000302:isolation & purification* / Q000502:physiology; D044162:Parathyroid Hormone-Related Protein; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification* / Q000502:physiology; D011994:Recombinant Proteins / Q000302:isolation & purification*; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis; D014162:Transfection
D009363:Neoplasm Proteins; C470121:PTHLH protein, human; D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000242:Cyclic AMP; D010959:Tissue Plasminogen Activator