Recombinant human prorenin from CHO cells: expression and purification.
Holzman TF,
Chung CC,
Edalji R,
Egan DA,
Gubbins EJ,
Rueter A,
Howard G,
Yang LK,
Pederson TM,
Krafft GA, et al.
Protein Biochemistry, Pharmaceutical Discovery Research, Abbott Laboratories, Illinois 60064.
The gene for human preprorenin was obtained from total RNA prepared from primary human chorion cells. An expression vector was constructed containing an SV40 early promoter, a human preprorenin cDNA, bovine growth hormone poly-A addition signal, and a dihydrofolate reductase (dhfr) expression cassette. This vector was inserted into the DXB-11 Chinese hamster ovary (CHO) cell line. The recombinant protein was exported by CHO cells into the tissue culture media. At harvest the prorenin levels ranged from approximately 1-5 mg/L. For prorenin isolation the cell culture supernatants were processed by filtration, concentration, dialysis, and batch extraction. Preparative-scale isolation of prorenin was accomplished using blue-dye chromatography and size-exclusion chromatography. The isolated prorenin yielded a single SDS-gel band with Mr approximately 40,000. The proprotein was characterized with respect to N-terminal sequence and N-linked sugar composition. Trypsin-activated renin prepared from the proprotein was characterized with respect to N-terminal sequence and pH-activity profile. Enzyme activity was measured with a newly developed fluorogenic peptide substrate containing the P6-P'3 sequence of human angiotensinogen.
PMID: 1963533 [PubMed - indexed for MEDLINE]