Preparation and characterization of a novel recombinant human parathyroid hormone (1-34) analog (Gly1-Gln26-rhPTH(1-34)) with enhanced biological activity.
A recombinant human parathyroid hormone (rhPTH) fragment (Gly1-Gln26-rhPTH(1-34)) which contains two amino acids substitutions (Gly1 and Gln26) was acquired through Escherichia coli expression system using a soluble fusion protein strategy. The soluble fusion protein MBP-Gly1-Gln26-rhPTH(1-34) was harvested after purification by Phenyl-Sepharose F.F and Q-Sepharose F.F chromatographies. Following tobacco etch virus (TEV) protease cleavage and further purification by SP-Sepharose F.F chromatography, 30.8 mg/L Gly1-Gln26-rhPTH(1-34) without tag was obtained with high purity up to 99%. Cyclic AMP (cAMP) stimulation assay suggested that Gly1-Gln26-rhPTH(1-34) could increase the biological activity by up to 13.89% and 6.34%. After daily subcutaneous injection (for 13 weeks) of 5, 10 and 20 microg of Gly1-Gln26-rhPTH(1-34)/1000g body weight, the mean Bone Material Density (BMD) of ovariectomized (OVXed) rats increased to 7.95-30.54% and 1.98-23.32%, compared to control-vehicle group (OVX, P<0.001) and sham- operated group (SHAM, P<0.01), respectively.
Xu
Xiao-Chao
XC
College of Life Sciences, Sichuan University, Chengdu, China.
Zhong
Shao-Dong
SD
Kai
Fan
F
Li
Ling-Rui
LR
Liu
Chao
C
Liu
Bo
B
Bao
Jin-Ku
JK
D000595:Amino Acid Sequence; D000818:Animals; D015519:Bone Density / Q000187:drug effects; D000242:Cyclic AMP / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005973:Glutamine / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D006801:Humans; D007279:Injections, Subcutaneous; D008969:Molecular Sequence Data; D010024:Osteoporosis / Q000188:drug therapy / Q000503:physiopathology; D010052:Ovariectomy; D010957:Plasmids / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D019379:Teriparatide / Q000737:chemistry / Q000494:pharmacology*
D011993:Recombinant Fusion Proteins; D005973:Glutamine; D019379:Teriparatide; D000242:Cyclic AMP; D010450:Endopeptidases; C099488:TEV protease; D005998:Glycine