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Expression, Purification and Characterization of Human Urodilatin in E. Coli

Expression, Purification and Characterization of Human Urodilatin in E. Coli

Ziyong Sun, Wei Lu, Yanchun Tang, Jing Zhang, Junyong Chen, Hualing Deng, Xuerong Li, Jian-Ning Liu

Protein Expr Purif. 2007 Oct;55(2):312-8.

PMID: 17544295

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP115966239	Urodilatin human		115966-23-9	Price

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