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Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli
Published on: August 3, 2017
Expression, purification and characterization of human urodilatin in E. coli
Ziyong Sun1, Wei Lu, Yanchun Tang
1Institute of Molecular Medicine and State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, 22 Hankou Road, Nanjing 210093, China.
Protein Expression and Purification
|June 5, 2007
Summary
This study developed a method to produce recombinant human urodilatin, a peptide hormone for treating heart failure. The process yielded highly pure and biologically active urodilatin, showing its potential for clinical applications.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Urodilatin is a kidney-synthesized peptide hormone regulating natriuresis and diuresis.
- It shows clinical promise for treating acute decompensated heart failure.
Purpose of the Study:
- To develop an efficient method for producing recombinant urodilatin in Escherichia coli.
- To characterize the biological activity of the produced urodilatin.
Main Methods:
- Cloning of a synthetic urodilatin gene into a pET32a vector for thioredoxin fusion protein expression in E. coli.
- Purification of the fusion protein using Ni-Sepharose affinity chromatography.
- On-column cleavage of urodilatin, followed by subtractive chromatography and reverse-phase HPLC.
Main Results:
- Overexpression of the thioredoxin-urodilatin fusion protein (Trx-urodilatin) reached 28% of total cell protein.
- High solubility (>85%) and purity (>97%) of the recombinant urodilatin were achieved.
- In vitro assays confirmed potent vasodilatory effects, comparable to synthetic standards (EC50 of (2.02+/-0.36)x10(-6)mg/ml).
Conclusions:
- A robust method for producing highly pure and active recombinant urodilatin was established.
- The yield is approximately 4.5mg per liter of E. coli culture.
- This recombinant production method holds significant potential for therapeutic applications in heart failure.
