title: Recombinant human prorenin from CHO cells: expression and purification.
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 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.
authors.affiliation: Protein Biochemistry, Pharmaceutical Discovery Research, Abbott Laboratories, Illinois 60064.
authors.forename: T F
authors.initials: TF
authors.lastname: Holzman
authors.forename: C C
authors.initials: CC
authors.lastname: Chung
authors.forename: R
authors.initials: R
authors.lastname: Edalji
authors.forename: D A
authors.initials: DA
authors.lastname: Egan
authors.forename: E J
authors.initials: EJ
authors.lastname: Gubbins
authors.forename: A
authors.initials: A
authors.lastname: Rueter
authors.forename: G
authors.initials: G
authors.lastname: Howard
authors.forename: L K
authors.initials: LK
authors.lastname: Yang
authors.forename: T M
authors.initials: TM
authors.lastname: Pederson
authors.forename: G A
authors.initials: GA
authors.lastname: Krafft
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004262:DNA Restriction Enzymes; D004792:Enzyme Precursors / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012083:Renin / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D014162:Transfection / Q000235:genetics
chemical_list: D004792:Enzyme Precursors; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D004262:DNA Restriction Enzymes; D012083:Renin