title: Expression, purification and characterization of human urodilatin in E. coli.
abstract: Urodilatin is a 32-amino acid peptide hormone synthesized in kidney to regulate natriuresis and diuresis. It has been shown clinically useful for the treatment of acute decompensated heart failure. A synthetic deoxyoligonucleotide encoding urodilatin was cloned into a pET32a vector immediately after the thioredoxin encoding sequence with a hexa-hisditine tag and an enterokinase recognition site incorporated in between. The fusion protein was overexpressed in Escherichia coli, which constituted 28% of the total cell proteins. More than 85% of Trx-urodilatin was soluble and purified nearly homogenous by Ni-Sepharose affinity chromatography. Urodilatin was then released from the fusion protein by the enterokinase treatment and separated from the fusion partner by the subtractive chromatography using Ni-Sepharose once again. The urodilatin sample was further purified with reverse phase HPLC. Via a biological activity assayed in vitro, it was found that urodilatin had a potent vasodilatory effect on rabbit aortic strips with an EC50 of (2.02+/-0.36)x10(-6)mg/ml, which was similar to that of the synthetic urodilatin standard. The method described here promises to produce about 4.5mg fully active recombinant urodilatin with homogeneity over 97% from one liter shaking flask culture of E. coli.
authors.affiliation: Institute of Molecular Medicine and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, 22 Hankou Road, Nanjing 210093, China.
authors.forename: Ziyong
authors.initials: Z
authors.lastname: Sun
authors.forename: Wei
authors.initials: W
authors.lastname: Lu
authors.forename: Yanchun
authors.initials: Y
authors.lastname: Tang
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
authors.forename: Junyong
authors.initials: J
authors.lastname: Chen
authors.forename: Hualing
authors.initials: H
authors.lastname: Deng
authors.forename: Xuerong
authors.initials: X
authors.lastname: Li
authors.forename: Jian-Ning
authors.initials: JN
authors.lastname: Liu
mesh_terms: D000595:Amino Acid Sequence; D009320:Atrial Natriuretic Factor / Q000235:genetics* / Q000302:isolation & purification*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003062:Codon; D004274:DNA, Recombinant; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification
chemical_list: D003062:Codon; D004274:DNA, Recombinant; D010446:Peptide Fragments; D011994:Recombinant Proteins; C056452:Ularitide; D009320:Atrial Natriuretic Factor