ABSTRACT
Human parathyroid hormone (hPTH) was highly expressed in Escherichia coli by inserted the synthesized whole hPTH cDNA into the vectors pBV220 and pET22b. After expression and disruption, the purified product was acquired through cation exchange chromatography and reverse phase chromatography. From the results of N-terminal sequencing and MALDI-TOF-MS analysis the recombiant prtein was indentified as intact hPTH. In in vitro Bioassays the recombinant hPTH stimulated adenylate cyclase as the standard did. In ovariectomized rats the recombinant hPTH markedly increased the femoral bone mass and bone mineral density.
Subject(s)
Animals , Female , Humans , Rats , Amino Acid Sequence , Base Sequence , Bone Density , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Genetics , Metabolism , Molecular Sequence Data , Ovariectomy , Parathyroid Hormone , Chemistry , Genetics , Metabolism , Pharmacology , Rats, Wistar , Sequence Alignment , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
N-carbamoylase is a part of hydantoinase operon which can transform N-carbamoylamino acid to corresponding ammo acids. The L-N-carbamoylase of Arthrobacter BT801, codied by the HyuC gene, is the rate-limiting and the only stereoselective enzyme. HyuC DNA fragment was amplified by PCR from the plasmid of pUC18-169. The target fragment was introduced into pPIC3. 5K plasmid to construct the pPIC3. 5K-hyuC expressing vector which was then transduced into Pichia pastoris GS115 cells after being linearized by BglⅡ digestion. Multi-copies insertion transformants were screened on G418 plates. The recombinant protein was proved to have biological activity of hydrolyzing N-carbamoylphenylalanine into phenylalanine through enzyme activity determination.