Recombinant human prorenin expression in baculovirus infected cells

Abstract

Renin is an aspartyl proteinase implicated in the regulation of blood pressure and fluid balance. As many other secreted enzymes, it is synthesized as a precursor, prorenin, that is cleaved by a processing enzyme during its migration through the cell sorting and secretion pathways. The prosegment plays an important role in the folding, the secretion and the inhibition if renin. A prediction of the three-dimensional structure of this zymogen has been derived from those of other aspartyl proteinases but recent findings have raised some doubts about the validity of this model. The best solution would be to crystallize the human prorenin but problems have been found with bacterial, yeast and mammalian expression systems. This study reports the expression of human prorenin in baculovirus infected arthropod cells and the optimization of this system. Up to 8 mg/L of recombinant human prorenin is produced by High-FiveTM cells. Comparison with prorenin produced by GH4 cells demonstrates that they have similar enzymatic activities and that they are recognized by the same antibody. However, the glycosylation of the recombinant prorenin produced in insect cells is different as it migrates at a lower molecular weight on a SDS-PAGE and it is not retained by an anion exchange column. By taking advantage of these different properties of the recombinant enzyme, a method for a large scale purification of prorenin has been designed