Stable expression, secretion, and characterization of active human renin in mammalian cells.
Human renin is synthesized as a 406-amino acid preprorenin protein that is processed by a signal peptidase during secretion, to release prorenin as a 386-amino acid zymogen. The 46-amino acid "pro" domain is removed by a renin-processing enzyme, to produce enzymatically active renin, by cleavage at an Arg-Leu bond. The effects of the renin-processing enzyme can be mimicked by trypsin activation, where high concentrations of trypsin are incubated with prorenin for brief periods of time, followed by excess trypsin inhibitor to minimize secondary proteolytic 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 deleted. This construct was introduced into mammalian cells and its expression was assayed in transient and stable systems. In COS-1 cells transfected with the prerenin expression vector pREN3, active renin was secreted with a specific activity of 1360 micrograms of angiotensin l/min/mg, compared with trypsin-activated prorenin, which has a specific activity of 818 micrograms 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 expressing the active form of renin was obtained by co-transfection of NRP cells with pREN3 and pHyg. A colony designated B/1 was identified, subcloned, and shown to secrete active renin (110 pg of renin/10(6) cells) optimally when maintained in both G418 and hygromycin.
Norman
J A
JA
Department of Cardiovascular Biochemistry, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000.
Hadjilambris
O
O
Baska
R
R
Sharp
D Y
DY
Kumar
R
R
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004247:DNA / Q000235:genetics; D004792:Enzyme Precursors / Q000235:genetics / Q000502:physiology; D015870:Gene Expression / Q000502:physiology; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D012083:Renin / Q000235:genetics* / Q000502:physiology; D014162:Transfection; D000092502:Renin Inhibitors
D004247:DNA; D004792:Enzyme Precursors; D009842:Oligopeptides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D012083:Renin; D000092502:Renin Inhibitors; C059981:SQ 32970