Aug 31, 1990

The purification and characterization of recombinant human renin expressed in the human kidney cell line 293

Biochemical and Biophysical Research Communications
C J VlahosB W Grinnell

Abstract

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.

Mentioned in this Paper

Shuttle Vectors
Transfection
HEK293 Cells
Proteins, Recombinant DNA
Cytokinesis of the Fertilized Ovum
Kidney
Trypsin
Enzyme Activity
Renin Measurement
Blood Enzyme Activity (Lab Test)

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