Expression and purification of human urodilatin by small ubiquitin-related modifier fusion in Escherichia coli.
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 microg/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.
Sun
Ziyong
Z
Institute of Molecular and Experimental Therapeutics, East China Normal University, 3663 ZhongShan North Road, Shanghai, 200062, China.
Xia
Zhinan
Z
Bi
Feng
F
Liu
Jian-Ning
JN
D000595:Amino Acid Sequence; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D010957:Plasmids; D015202:Protein Engineering; D011499:Protein Processing, Post-Translational; D020673:Protein Renaturation; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D012995:Solubility
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; C056452:Ularitide; D009320:Atrial Natriuretic Factor; D010447:Peptide Hydrolases