Expression, purification and characterization of human urodilatin in E. coli.
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.
Sun
Ziyong
Z
Institute of Molecular Medicine and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, 22 Hankou Road, Nanjing 210093, China.
Lu
Wei
W
Tang
Yanchun
Y
Zhang
Jing
J
Chen
Junyong
J
Deng
Hualing
H
Li
Xuerong
X
Liu
Jian-Ning
JN
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
D003062:Codon; D004274:DNA, Recombinant; D010446:Peptide Fragments; D011994:Recombinant Proteins; C056452:Ularitide; D009320:Atrial Natriuretic Factor