Suppression of the degradation of recombinant human apolipoprotein E by a protease inhibitor obtained from fetal bovine serum in serum-free culture.
The recombinant human apolipoprotein E (Apo-E) produced by Chinese hamster ovary cells (CHO-322 cells) in serum free culture was degraded to 24K and 23K fragments that contained N-terminal amino acid. The degradation site of Apo-E to 24K fragment was between Arg180 and Leu181 and the C-terminal amino acid of 23K fragment was Gly169. In fetal bovine serum (FBS)-containing culture, the degradation was inhibited. However, in calf serum (CS) the inhibitory activity was not detected. Thus, we attempted the purification of the factor with this inhibitory activity from FBS. A protease inhibitor was purified to give a single peak from FBS by ammonium sulfate precipitation and combination of several column chromatographies. When this FBS-derived protease inhibitor (FBS-d-PI) was added to serum-free culture of CHO-322 cells, degradation of recombinant Apo-E to the 24K and 23K fragments was dose-dependently suppressed and accumulation of intact Apo-E in culture supernatant was observed. FBS-d-PI was found to be a glycoprotein with relative molecular size of 75K daltons under reducing condition, and 85K daltons under nonreducing condition by SDS-PAGE. A complex of FBS-d-PI and a cellular protease was also detected in culture supernatant by western blot analysis using mouse monoclonal antibodies against FBS-d-PI.
Shimomura
T
T
Biosciences Laboratory, Mitsubishi Kasei Corp., Yokohama, Japan.
Honda
T
T
Oouchi
C
C
Kondo
J
J
Nagaike
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001057:Apolipoproteins E / Q000096:biosynthesis*; D001769:Blood; D015153:Blotting, Western; D002460:Cell Line; D006224:Cricetinae; D003470:Culture Media / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012710:Serum Albumin, Bovine / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*
D001057:Apolipoproteins E; D003470:Culture Media; D046911:Macromolecular Substances; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D012710:Serum Albumin, Bovine