Expression and characterization of recombinant rat parathyroid hormone-related peptide (1-141) and an amino-terminally-truncated analogue (38-141).
We have synthesized and purified recombinant parathyroid hormone related peptide (PTHrP (1-141)) and PTHrP (38-141) using an E. coli system that requires minimal purification. The cDNAs encoding PTHrP (1-141) and PTHrP (35-141) respectively were inserted into the multiple cloning site of the pTrcHis-B bacterial expression plasmid. The PTHrP encoded sequences were thereby fused at their NH2-termini to six histidine residues within the fusion protein. The recombinant plasmids were transfected into E. coli cells and PTHrP synthesis was induced by addition of 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG) at 37 degrees C. The recombinant fusion proteins were purified by binding of the histidine residues to a nickel column followed by gradient elusion and dialysis. PTHrP (1-141) was released from its fusion protein by cyanogen bromide cleavage, whereas PTHrP (38-141) was released by enzymatic digestion with enterokinase. This rapid isolation method resulted in pure PTHrP (1-141) and (38-141) as judged by SDS-polyacrylamide gel electrophoresis and NH2-terminal sequence analysis. PTHrP (1-141) stimulated cAMP accumulation and mobilized intracellular calcium ([Ca2+]i) in UMR106 osteoblast-like cells, and stimulated phosphate transport in OK/E renal cells, whereas PTHrP (38-141) was inert in these bioassays. Availability of PTHrP and its NH2-terminally truncated analogue, which lacks the sequence necessary for its hypercalcemic actions, will enable their biological activities to be examined in greater detail.
Rabbani
S A
SA
Department of Medicine, McGill University, Royal Victoria Hospital, Montreal, Quebec, Canada.
Harakidas
P
P
Gladu
J
J
Srivastava
S
S
Diarra
A
A
Sauvé
R
R
Karaplis
A C
AC
Henderson
J E
JE
Goltzman
D
D
Hendy
G N
GN
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D017136:Ion Transport / Q000187:drug effects; D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism; D011506:Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology
D044162:Parathyroid Hormone-Related Protein; D010446:Peptide Fragments; D010710:Phosphates; D011506:Proteins; D011993:Recombinant Fusion Proteins; C108117:parathyroid hormone-related peptide (38-141); C066078:parathyroid hormone-related protein (1-141); D000242:Cyclic AMP; D002118:Calcium