STABLE EXPRESSION, SECRETION, AND CHARACTERIZATION OF ACTIVE HUMAN RENIN IN MAMMALIAN-CELLS

Human renin is synthesized as a 406-amino acid preprorenin protein tha t is processed by a signal peptidase during secretion, to release pror enin as a 386-amino acid zymogen. The 46-amino acid "pro" domain is re moved by a renin-processing enzyme, to produce enzymatically active re nin, by cleavage at an Arg-Leu bond. The effects of the renin-processi ng enzyme can be mimicked by trypsin activation, where high concentrat ions of trypsin are incubated with prorenin for brief periods of time, followed by excess trypsin inhibitor to minimize secondary proteolyti c processing by trypsin. In order to study the role of the pro segment in the secretion, folding, and activity of human renin, we engineered a construct where the pro domain from the preprorenin cDNA was delete d. This construct was introduced into mammalian cells and its expressi on was assayed in transient and stable systems. In COS-1 cells transfe cted with the prerenin expression vector pREN3, active renin was secre ted with a specific activity of 1360 mu-g of angiotensin l/min/mg, com pared with trypsin-activated prorenin, which has a specific activity o f 818-mu-g of angiotensin l/min/mg. The active renin secreted in this system had a significantly reduced potency for the renin inhibitor SQ 32,970. These results demonstrate that the pro segment is dispensable for the folding and secretion of renin. A permanent cell line expressi ng the active form of renin was obtained by co-transfection of NRP cel ls with pREN3 and pHyg. A colony designated B/1 was identified, subclo ned, and shown to secrete active renin (110 pg of renin/10(6) cells) o ptimally when maintained in both G418 and hygromycin.