Recombinant human prorenin from CHO cells: expression and purification.
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.
Holzman
T F
TF
Protein Biochemistry, Pharmaceutical Discovery Research, Abbott Laboratories, Illinois 60064.
Chung
C C
CC
Edalji
R
R
Egan
D A
DA
Gubbins
E J
EJ
Rueter
A
A
Howard
G
G
Yang
L K
LK
Pederson
T M
TM
Krafft
G A
GA
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
D004792:Enzyme Precursors; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D004262:DNA Restriction Enzymes; D012083:Renin