Expression and purification of human urodilatin by small ubiquitin-related modifier fusion in Escherichia coli |
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Authors: | Ziyong Sun Zhinan Xia Feng Bi Jian-Ning Liu |
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Institution: | (1) Institute of Molecular and Experimental Therapeutics, East China Normal University, 3663 ZhongShan North Road, Shanghai, 200062, China |
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Abstract: | To prevent in vivo degradation, small peptides are usually expressed in fusion proteins from which target peptides can be
released by proteolytic or chemical reagents. In this report, small ubiquitin-related modifier (SUMO) linked with a hexa-histidine
tag was used as a fusion partner for the production of recombinant human urodilatin, a hormone for the treatment of acute
decompensated heart failure. The fusion protein, which was overexpressed mainly as inclusion bodies in Escherichia coli, constituted about 25% of the total cell proteins. After purification by Ni-sepharose affinity chromatography and renaturation
in refolding buffer, the fusion protein was cleaved with SUMO protease 1. Urodilatin was separated from the fusion partner
by the subtractive chromatography using Ni-sepharose once again, and then further purified with reverse-phase high performance
liquid chromatography. In vitro activity assay demonstrated that the recombinant urodilatin had a potent vasodilatory effect
on rabbit aortic strips with an EC50 of 1.77 ± 0.53 μg/ml, which was similar to that of the synthetic urodilatin standard. The expression strategy presented in
this study allows convenient high yield and easy purification of small recombinant peptides with native sequences.
Z. Sun and Z. Xia contribute equally to the work. |
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Keywords: | Small ubiquitin-related modifier SUMO protease 1 Urodilatin Cleavage Fusion expression |
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