Study on preparation and activity of a novel recombinant human parathyroid hormone(1-34) analog with N-terminal Pro-Pro extension.
A recombinant human parathyroid hormone fragment, Pro-Pro-hPTH(1-34), with molecular weight of 4311.46 was acquired through gene engineering. It was then isolated and purified. The homogeneity of this fragment was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), high performance liquid chromatography(HPLC), isoelectronic focusing (IEF) electrophoresis and mass spectrometry(MS) methods. Its isoelectric point is 8.0 which was determined by IEF. It was found that the hormone fragment significantly induced calcium increment as compared to the control group (P<0.001) in Parsons's Chicken Assay, an established bioassay for the evaluation of the PTH effect. After the 3-month-old ovariectomized (OVXed) rats, the OVXed rat is one of the two models required by the U.S. Food and Drug Administration for the preclinical assessment of drugs for treating osteoporosis [DeLuca PP, Dani BA. Skeletal effects of parathyroid hormone (1-34) in ovariectomized rats with or without concurrent administration of salmon calcitonin. Am Assoc Pharm Sci 2001;3(4):E27 [1]]. Sprague-Dawley rats were fed for 14 weeks, daily subcutaneous injections of Pro-Pro-hPTH(1-34) for 16 weeks (0.4, 0.6 or 0.9 nmol/100 g body weight), reduced the ovariectomy (OVX)-triggered mass loss of vertebral trabecular bone. The mean Bone Material Density (BMD) increased to 29.2-34.5% in 3-month-old OVXed rats compared to control-vehicle group (P<0.001) and increased to 17.5-22.3% compared to sham-operated groups (P<0.01). In short, A recombinant Pro-Pro-hPTH(1-34) was harvested in purified form and its physico-chemical characterization was determined. It showed significantly enhanced activity upon two typical models for PTH fragments. It can increase the mineral density of vertebral trabecular bone just as synthetic hPTH(1-34), and the functional activity of Pro-Pro-hPTH(1-34) should be due to the removing of Pro-Pro- by Dipeptidyl peptidase IV (DPPIV). This study opened out a simplified method which was cheaper, faster than the conventional one for producing active hPTH fragment, and its applied prospect would be good; Furthermore, it may open up our own path in finding new methods for post-processing of gene-engineering product.
Chunxiao
Wang
W
Laboratory of Minigene Pharmacy, School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China. cxwang@shfu.edu.cn
Jingjing
Liu
L
Yire
Xiao
X
Min
Ding
D
Zhaohui
Wang
W
Gaofu
Qi
Q
Xiangchun
Shen
S
Xuejun
Wang
W
Jie
Wu
W
Taiming
Li
L
D000818:Animals; D001681:Biological Assay; D015519:Bone Density / Q000187:drug effects; D002116:Calcitonin / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D004151:Dipeptides / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005818:Genetic Engineering; D005822:Genetic Vectors; D006801:Humans; D007279:Injections, Subcutaneous; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D008159:Lumbar Vertebrae / Q000378:metabolism; D013058:Mass Spectrometry; D008970:Molecular Weight; D010052:Ovariectomy; D010281:Parathyroid Hormone / Q000008:administration & dosage / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D017422:Sequence Analysis, DNA; D013997:Time Factors
D004151:Dipeptides; C470125:PTH protein, human; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C448039:prolyl-proline; D002116:Calcitonin; D002118:Calcium