Purification and characterization of human renalase produced in Escherichia coli as a FAD-containing flavoprotein

Renalase (1) is a novel protein, ubiquitous in vertebrates, and expressed in various tissues and organs, including plasma, kidney, heart, and nervous system. Renalase has been shown to be secreted by the kidney into the bloodstream, where it reduces blood pressure and heart rate. Decrease of renalase plasma levels in patients suffering from chronic kidney diseases could represent a key factor in favoring cardiovascular complications. Renalase and monoamine oxidases (MAOs) share a low degree of sequence identity (about 17%). This similarity led to the hypothesis that renalase could be a MAO-like flavin-containing enzyme. Despite its potential relevance for human health, the biochemical characterization of renalase is still lacking, possibly due to difficulties in obtaining it in recombinant form. By expressing two different gene constructs, we found that human renalase is mainly produced in E. coli as inclusion bodies. However, by carefully choosing the host strain and optimizing the expression conditions, significant amounts of soluble products were obtained. The proteins have been purified to homogeneity exploiting their N-terminal His-tag. Linking of renalase to the SUMO protein did not improve solubility, but yielded untagged renalase after proteolytic processing of the fusion product. The procedures here reported for the production and isolation of renalase yielded milligrams amounts of highly pure recombinant protein. The two renalase forms displayed the same molecular properties. They bind equimolar amounts of non-covalently bound FAD and appear to be correctly folded by various criteria. They were found to possess the in vivo activity on blood pressure previously described for the human protein. (1) G.V. Desir, Kidney Int. 76 (2009) 366-370.