کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
2021298 | 1069239 | 2007 | 7 صفحه PDF | دانلود رایگان |
![عکس صفحه اول مقاله: Expression, purification and characterization of human urodilatin in E. coli Expression, purification and characterization of human urodilatin in E. coli](/preview/png/2021298.png)
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) × 10−6 mg/ml, which was similar to that of the synthetic urodilatin standard. The method described here promises to produce about 4.5 mg fully active recombinant urodilatin with homogeneity over 97% from one liter shaking flask culture of E. coli.
Journal: Protein Expression and Purification - Volume 55, Issue 2, October 2007, Pages 312–318