Purification and characterization of recombinant human renin for X-ray crystallization studies.
BACKGROUND
The renin-angiotensin-aldosterone system (RAS) cascade is a major target for the clinical management of hypertension. Although inhibitors of various components of this cascade have been developed successfully, development of renin inhibitors has proven to be problematic. The development of these inhibitors has been hindered by poor bioavailability and complex synthesis. However, despite the challenges of designing renin inhibitors, the enzyme remains a promising target for the development of novel treatments for hypertension. X-ray crystallographic data could greatly assist the design and development of these inhibitors. Here we describe the purification and characterization of recombinant human renin for x-ray crystallization studies.


RESULTS
A cDNA encoding the full length of native human preprorenin (406 amino acid residues) was introduced into the HEK-293 cell line. A clonal cell line expressing prorenin was generated and grown under serum free conditions in a hollow fiber bioreactor. Prorenin was constitutively secreted and purified directly from the conditioned medium. Concanavalin A chromatography effectively enriched and purified prorenin to 90% homogeneity in a single step. Prorenin was converted to active renin by trypsin digestion to remove the propeptide. Active renin was further purified using a cation exchange column followed by a gel filtration column. Biochemical characterization of the recombinant enzyme showed both binding and catalytic properties were essentially identical to previously reported activities for purified renin. Crystals were grown using this material in our X-ray structure studies, and high resolution diffraction was obtained.


CONCLUSIONS
This present work describes a simple and efficient method for the generation and purification of active human renin. The protein is highly pure and is suitable for supporting structural biology efforts.
Wu
Zhongren
Z
Vitae Pharmaceuticals Inc., Discovery Biology, Fort Washington, PA, 19034, USA. zwu@vitaerx.com
Cappiello
Maria G
MG
Scott
Boyd B
BB
Bukhtiyarov
Yuri
Y
McGeehan
Gerard M
GM
D000595:Amino Acid Sequence; D002460:Cell Line; D003460:Crystallization; D018360:Crystallography, X-Ray; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D015394:Molecular Structure; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D012083:Renin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D011994:Recombinant Proteins; D012083:Renin