Recombinant human prorenin from CHO cells: Expression and purification |
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Authors: | Thomas F. Holzman, Christine C. Chung, Rohinton Edalji, David A. Egan, Earl J. Gubbins, Annemarie Rueter, Gail Howard, Lana K. Yang, Terry M. Pederson Grant A. Krafft etAlia" >,et al. |
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Affiliation: | (1) Protein Biochemistry, Pharmaceutical Discovery Research, Pharmaceutical Products Division, Abbott Laboratories, 60064 Abbott Park, Illinois;(2) Molecular Biology, Pharmaceutical Discovery Research, Pharmaceutical Products Division, Abbott Laboratories, 60064 Abbott Park, Illinois;(3) Probe Molecular Design, Abbott Diagnostics Division, Abbott Laboratories, 60064 Abbott Park, Illinois |
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Abstract: | 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 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 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 andpH-activity profile. Enzyme activity was measured with a newly developed fluorogenic peptide substrate containing the P6-P3 sequence of human angiotensinogen. |
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Keywords: | Prorenin renin N-terminal sequence fluorogenic substrate cDNA cloning |
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