The purification and characterization of recombinant human renin expressed in the human kidney cell line 293.
The cDNA encoding human preprorenin has been introduced into the adenovirus-transformed human kidney cell line 293. The recombinant 293 cells expressed and secreted prorenin; trypsin was used to activate the secreted prorenin to renin in vitro. The recombinant protein was purified to homogeneity by a single affinity chromatographic step. Using synthetic tetradecapeptide, the Km was 57.1 +/- 9.3 microM and the kcat was (7.48 +/- 1.57) x 10(3)/hr. Activation with trypsin resulted in a secondary cleavage between Arg53 and Leu54 generating a two chain form held together via a disulfide between Cys51 and Cys58. This secondary cleavage did not affect enzyme activity as determined by the ability of renin to degrade a synthetic tetradecapeptide substrate. Our paper demonstrates the potential for producing large quantities of renin from human kidney cells and also suggests that the use of trypsin, which has been widely used to convert prorenin to renin in vitro, causes a secondary cleavage in the renin peptide chain.
Vlahos
C J
CJ
Lilly Research Laboratories, Indianapolis, IN 46285.
Walls
J D
JD
Berg
D T
DT
Grinnell
B W
BW
D000595:Amino Acid Sequence; D002460:Cell Line; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000302:isolation & purification; D012083:Renin / Q000302:isolation & purification* / Q000378:metabolism; D014162:Transfection
D004792:Enzyme Precursors; D011994:Recombinant Proteins; D012083:Renin