Isolation and characterization of native human renin derived from Chinese hamster ovary cells.
Transfection of Chinese hamster ovary (CHO) cells with a plasmid containing the cDNA for human preprorenin has provided cell lines that secrete 15 mg of native prorenin per liter of culture medium. Tryptic activation of the prorenin occurs by selective cleavage of the Arg66-Leu67 bond (numbering as in preprorenin). The renin product, purified in a single step and in high yield by affinity chromatography, is fully stable for as long as 8 months when stored in solution at 4 degrees C and pH 6.5. Purity of the renin was judged to be greater than 95% by gel electrophoresis, compositional and N-terminal sequence analyses, and specific enzyme activity. An important aspect of the present work is the development of a direct assay for renin which permits accurate and reproducible evaluation of enzyme units and kinetic parameters. Application of methods described herein, combined with appropriate scale-up fermentation capabilities, provides the means for generating gram quantities of human renin and its zymogen.
Poorman
R A
RA
Department of Molecular Biology, Upjohn Company, Kalamazoo, Michigan 49001.
Palermo
D P
DP
Post
L E
LE
Murakami
K
K
Kinner
J H
JH
Smith
C W
CW
Reardon
I
I
Heinrikson
R L
RL
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics; D004792:Enzyme Precursors / Q000235:genetics; D005260:Female; D006168:Guinea Pigs; D006801:Humans; D010053:Ovary; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012083:Renin / Q000235:genetics / Q000302:isolation & purification*; D014162:Transfection
D004792:Enzyme Precursors; D011994:Recombinant Proteins; D004247:DNA; D012083:Renin